Effects of commonly used disinfectants on bacterial load,hatchability and survival of Bluefin Sea bream (Sparidentex hasta) eggs

The efficacy of four chemical reagents, iodophor, formalin, hydrogen peroxide and bronopol as fish egg surface disinfectants were evaluated in bluefin sea bream (Sparidentex hasta). Fertilized eggs were counted and subjected to a static bath dip treatment in different concentrations of the above chemicals for 4 min before being incubated at 20 ± 0.5°C for 40 h. Treatment efficacy of the different disinfectants was evaluated by assessing the bactericidal activity, egg hatch percentage and survival of larvae up to 3 days post hatch. Results showed that iodophor at medium concentrations (75 and 100 ppm) was the best of all tested disinfectants in bacterial killing ability (12% reduction in the bacterial counts), egg hatching per cent (99.8% and 99.6% respectively) and larval survival up to 3 days post hatch (50.8% and 54.8% respectively). Formalin was the second best disinfectant at levels of 100 and 150 ppm. Hydrogen peroxide gave good results compared with the control while, bronopol showed discouraging results. In conclusion, iodophor appeared to be suitable for bluefin sea bream eggs disinfection with a 4 min exposure to 75–100 ppm when applied 14–16 h after egg fertilization.     

Disinfection of Sparus aurata eggs with glutaraldehyde

This study was conducted to determine suitable conditions fordisinfecting eggs of gilthead sea bream (Sparus aurata)with glutaraldehyde. Effects of the developmental stage (4–8 cells,morula, blastopore closure or heart beating), of the concentration (C = 200,300or 400 ppm) and of the duration of the glutaraldehyde treatment (T= 2 to 10 min) were investigated. Before the blastopore closurestage, egg manipulation and treatment induced mortality. After this stage, thetoxicity of the glutaraldehyde treatment was negligible if the value of theproduct C × T was less than 1000. Above this value, the percentage ofhatching and of normal larvae decreased and the percentage of imprisoned larvaeincreased. Toxic effects of glutaraldehyde varied according to the egg qualityat the time of the treatment. It was concluded that 200 ppmglutaraldehyde for 4 min, at the blastopore closure stage or attheheart beating stage, were acceptable conditions for disinfecting gilthead seabream eggs at 18 °C.     

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.     

Effects of different bronopol treatments on final survival rates in the artificial incubation of crayfish eggs (Pacifastacus leniusculus,Astacidae)

Two experiments were conducted on the effects of bronopol in the artificial incubation of crayfish eggs (Pacifastacus leniusculus) with the aim to search an alternative to formaldehyde. In the first experiment, 50, 250, 500 and 1000 ppm bronopol and 3000 ppm formaldehyde (control) in periodical administrations were tested on a density of 6.6 eggs cm^?2. After 44 days of incubation, the highest survival was obtained with 1000 ppm bronopol (81.9% to stage 2 juvenile, with no significant difference from formaldehyde), whereas lower bronopol concentrations resulted in significantly lower survival. In the second experiment, 1000, 3000 and 5000 ppm bronopol and 3000 ppm formaldehyde (control) administered for 15 min every second day were tested on eggs at a density of 20 eggs cm^?2. After 78 days of incubation, bronopol at 3000 ppm allowed for a stage 2 juvenile survival rate of 65.0% (with no significant difference from formaldehyde), whereas significantly lower survival was obtained with 1000 ppm or 5000 ppm. This study shows that bronopol may constitute an alternative to formaldehyde in the artificial incubation of crayfish eggs. A concentration of 3000 ppm administered for 15 min every second day may be adequate even on long incubations at high densities (at least 20 eggs cm^?2, one complete layer).     

Effects of storage and incubation temperature on the viability of eggs, embryos and larvae in two strains of an African catfish, Heterobranchus longifilis (Siluriformes, Clariidae)

Two experiments, dealing with short‐term storage of ova and thermal conditions to optimize gamete and eggs management in hatcheries of the African catfish, Heterobranchus longifilis, were carried out. In the first experiment, ova collected by stripping from two strains of H. longifilis were stored for intervals up to 8 h at two temperature regimes: in a domestic refrigerator (3–5°C) and at ambient room temperature (20.5–22°C). In the second experiment, eggs were incubated from fertilization to hatching at different experimental temperatures (21, 25, 29, 32 and 35°C) to determine the effects of temperature on the kinetics of white egg appearance, hatching times and hatching quality. Gamete storage at warmer temperatures significantly prolonged viability irrespective of the strain used. In fact, the hatching rate for ova stored at 20.5–22 and 3–5°C for 5 h ranged between 75.2–79.3% and 6.5–9.4% respectively. Loss of viability was most noticeable after 6 h storage at ambient room temperature. Post‐storage viability significantly declined after 2 h exposure to the domestic refrigerator temperature. No hatching of normal larvae took place after 8 h post‐storage time. Results from the second experiment showed that time to maximum whitening of eggs was both strain‐ and temperature‐dependent. The time to maximum mortality of eggs was shorter in the Layo strain (LS) than in the Noun strain (NS), regardless of incubation temperature. The appearance of white eggs was shorter with increasing incubation temperatures. Hatching times decreased with increasing temperature, regardless of strain. Hatching took place from 21 to 27 h and 19 to 24 h after fertilization at temperature of 29°C, respectively, for NS and LS. The length of the hatching period was remarkably shorter for LS than NS at any tested incubation temperature, except 35°C. No hatching took place at 21°C. The highest proportion of normal larvae occurred at 25 and 29°C, respectively, for NS and LS. Hatching rate was highest at 25 and 29°C, respectively, for NS and LS. There was a significantly higher proportion of deformed larvae at 35°C regardless of the strain.     

Effect of incubation temperature on the embryonic development and yolk‐sac larvae of the Pacific red snapper Lutjanus peru (Nichols & Murphy, 1922)

The effect of incubation temperature on embryonic development and yolk‐sac larva of the Pacific red snapper Lutjanus peru were evaluated by testing the effect of 26, 28 and 30°C, as this is the natural thermal interval reported during the spawning season of Pacific red snapper in the Gulf of California, Mexico. Sixteen developmental stages were observed. The incubation temperature affected the rate of development and time to hatching, being shorter at 30 than at 26°C, but no significant effect (P < 0.05) on larval length at hatching was registered. The depletion rate of yolk sac and oil globule was affected by incubation temperature particularly during the first 12 h post hatching (hph). At the end of the experiment (48 hph), significantly (P < 0.05) larger larvae were recorded at 26°C (TL = 3.22 ± 0.01 mm) than at 28° (TL = 3.01 ± 0.02 mm) and 30°C (TL = 2.97 ± 0.05 mm). Incubation of newly fertilized eggs at 26°C produces larger larvae, which may help to improve feeding efficiency and survival during first feeding.     

Chemical surface disinfection of eggs of Baltic cod,Gadus morhua L.

The effect of two disinfectants on eggs and larvae of Baltic cod, Gadus morhua, was investigated. The eggs were disinfected for 10 min using various concentrations of either glutaraldehyde (100, 200, 400, 600 and 800 mg L^?1) or iodophor (10, 50, 100 and 150 mg L^?1), 1–4‐days post‐fertilization. Bactericidal effect of disinfection, survival to hatching, hatching success and larval abnormalities were assessed. Larval survival was recorded at 5‐, 10‐ and 15‐days post‐hatch (dph). Although Baltic cod eggs have an unusually thin chorion, they could tolerate surface disinfection. A reduction in bacterial growth was observed with increased concentrations of disinfectant (3.0 × 10⁷–1.6 × 10¹ CFU mL^?1). Abnormalities in newly hatched larvae were not related to disinfection. Survival of the yolk sac larvae was significantly better for eggs treated with 400 mg L^?1 glutaraldehyde for 10 min at 10 and 15 dph. Effective disinfection was also recorded using 100 mg L^?1 Actomar K30. Egg batch effect rather than initial bacterial concentration, disinfectant type or incubation method determined the survival of the eggs to hatching and survival of larvae. Because of the carcinogenic effect of glutaraldehyde, iodophor is recommended for routine disinfection of cod eggs.     

Effects of different step‐wise temperature increment regimes during egg incubation of Atlantic cod (Gadus morhua L.) on egg viability and newly hatched larval quality

We carried out an experiment to determine how rapidly the early incubation temperature of Atlantic cod eggs can be increased without affecting normal embryonic development and hatching. Atlantic cod eggs were incubated at a constant low temperature (4.5 ± 0.5°C; T5 – control) and four temperature increment treatments where the temperatures were increased stepwise from 4.5°C at zygote stage to 9.5 ± 05°C (T1‐8 h, T2‐32 h, T3‐64 h and T4‐96 h). Embryonic cell symmetry, embryonic mortality, hatching success and larval skeletal abnormalities, length and yolk sac volume were recorded. Larval samples were also taken at hatch for histological analysis. Except for higher egg mortality and lower hatching success in the T1, the differences among experimental groups were minor. Cell asymmetries and embryo mortalities were not significantly different between the control and T2–T4 treatment groups. Control larvae were significantly longer and had smaller yolk reserves at hatch than T1–T4 larvae and larvae from T2 had the largest yolk reserves. Tissue and organ histology of hatched larvae were similar. Considering embryonic cleavage pattern, hatching success and larval morphology and histology, a gradual increment of temperature in 32 h seems to be the better choice for future developmental programming studies in Atlantic cod.     

Disinfection of Sparus aurata eggs with glutaraldehyde

This study was conducted to determine suitable conditions fordisinfecting eggs of gilthead sea bream (Sparus aurata)with glutaraldehyde. Effects of the developmental stage (4–8 cells,morula, blastopore closure or heart beating), of the concentration (C = 200,300or 400 ppm) and of the duration of the glutaraldehyde treatment (T= 2 to 10 min) were investigated. Before the blastopore closurestage, egg manipulation and treatment induced mortality. After this stage, thetoxicity of the glutaraldehyde treatment was negligible if the value of theproduct C × T was less than 1000. Above this value, the percentage ofhatching and of normal larvae decreased and the percentage of imprisoned larvaeincreased. Toxic effects of glutaraldehyde varied according to the egg qualityat the time of the treatment. It was concluded that 200 ppmglutaraldehyde for 4 min, at the blastopore closure stage or attheheart beating stage, were acceptable conditions for disinfecting gilthead seabream eggs at 18 °C.     

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.     

Investigations on the effect of formalin and iodophor on embryo and larvae development in pikeperch,Sander lucioperca

ABSTRACT

An experiment was conducted to evaluate the effects of incubating pikeperch, Sander lucioperca, eggs in formalin and iodophor solutions for 15 min on embryo survival, the hatching rate, as well as on the rate of misshaped larvae, in order to develop methods for egg surface disinfection. Embryos in the morula stage, in the epiboly stage, and at the beginning of heart beat and blood circulation tolerated formalin concentrations up to 1,500 ppm for 15 min. However, they were very susceptible to iodophor treatment, as >0.1% iodophor solution (=13 ppm active iodine) significantly decreased the percentage of ready-to-hatch embryos and the percentage of hatched larvae. These data of this study recommend the use of formalin at a concentration of up to 1,500 ppm to disinfect pikeperch eggs.     

Antifungal effect of Origanum onites essential oil as an alternative to formalin in the artificial incubation of narrow‐clawed crayfish (Astacus leptodactylus Eschscholtz, 1823)

As alternative to formalin, the antifungal effect of a plant product [Origanum onites L. (Lamiaceae) oil] was investigated for use in the artificial incubation of narrow‐clawed crayfish (Astacus leptodactylus Eschscholtz) eggs. For this purpose, this study was conducted as two experiments. In experiment I, the eggs were artificially incubated for 40 days. In experiment II, juveniles were cultured to determine effects of O. onites oil on juveniles for 30 days. The experimental groups were as follows: formalin (3500 ppm for 15 min), O. onites oil (300 ppm for 15 min, 700 ppm for 2 min and 1000 ppm as a dip treatment 15 split‐second) and a control (no treatment). In the experiment I, the highest hatching rate (86%) and survival rate of stage II juveniles (80%) were observed in 1000 ppm dip group. These results were similar to that of formalin group (85% and 79%) respectively. The control group exhibited the lowest hatching rate (49%) and stage II rate (42%) compared with the 1000 ppm dip group and 3500 ppm formalin treatments. However, other concentrations (300 and 700 ppm) of O. onites showed toxic effects on the eggs and there was no hatching. In the experiment II, the survival rate and growth performance of the crayfish juveniles were similar in all groups. This study indicated that the 1000 ppm O. onites dip treatment could be a good alternative to formalin for improved egg hatchability in the artificial incubation of crayfish eggs.     

Tetraploid induction in tropical oysters,Crassostrea belcheri (Sowerby) and Crassostrea iredalei (Faustino)

Tetraploid induction has been conducted on temperate oysters but not on tropical oysters. In this study, different heat shocks (32, 35 and 38°C) and cold shocks (1, 4 and 7°C) were used to induce tetraploidy in two tropical oyster species, Crassostrea belcheri and Crassostrea iredalei, through meiosis I inhibition. Temperature shocks were applied on the newly fertilized eggs at 8–10 min post fertilization and terminated when second polar bodies began to form in the control eggs. The ploidy of the larvae and spat was determined via direct chromosome count. The percentage of larval survival until Day 20 was low (between 0.4% and 42.9%) for both temperature shocks and oyster species. No surviving larva was recorded for induction at 1, 4 and 38°C. Tetraploid spat was only recorded in C. iredalei but the percentage is low through heat shock induction of 32 and 35°C. This study shows that the tetraploid induction success rate was slightly higher in C. iredalei compared to C. belcheri. No surviving tetraploid spat were recorded for both oyster species through the cold shock method. This study shows that heat shock can be used to inhibit meiosis for the production of tetraploids but more experiments need to be conducted to determine the optimum temperature when dealing with tropical oysters.     

Development of embryos and larvae in the common Japanese conger Conger myriaster

Development of embryos and larvae in the common Japanese conger Conger myriaster was observed after artificial fertilization. Eggs were obtained from females matured artificially by hormone injections and milt was obtained from males matured naturally. Fertilized eggs were kept in seawater at 12–14°C. The first cleavage occurred at 4 h, epiboly began at 24 h, the embryonic body was formed at 38 h and hatching occurred at 84 h after insemination. Newly hatched larvae were approximately 2.5 mm (total length) and similar to those of Anguilla japonica in terms of external features. The mouth and anus opened on the 7th day after hatching. Pigments began to appear at the tip of the tail on the 10th day. The total length of the larvae reached approximately 8 mm on the 11th day. Eye pigmentation began on the 14th day. One larva lived for 19 days without food.     

Disinfection of almaco jack (Seriola rivoliana Valenciennes) eggs: Evaluation of three chemicals

Almaco jack (Seriola rivoliana Valenciennes) is an excellent candidate for aquaculture due to its fast growth rate and high market value. While S. rivoliana have adapted well to captivity, survival at early life stages can be improved to increase profitability during production. A wide range of variables cause larval mortalities but high bacterial loads in rearing tanks are often correlated with these losses. The aim of this study was to investigate the effect of egg disinfection on bacterial load and hatch rate of S. rivoliana. Disinfectants tested included formalin (F100 and F200; 100 and 200 mg/L, respectively, for 60 min), hydrogen peroxide (HPO; 300 mg/L for 10 min) and peracetic acid/hydrogen peroxide (PAA/HPO; 15.7 mg/L/39.6 mg/L for 1 min). Concentrations and contact times were determined based on current use in marine aquaculture and preliminary trials. Eggs treated with HPO and F100 had significantly higher hatch rates than the untreated control group. All treatments significantly decreased total Vibrio counts compared to untreated eggs; however, total bacterial counts were only decreased following treatments with PAA/HPO and F200. To prevent egg mortality due to bacterial overgrowth, consideration should be given to the use of surface disinfection using HPO or F100. Future studies should investigate the use of peracetic‐based products at lower doses.     

Effects of light and short-term temperature elevation on the 48-h hatching success of cold-stored Acartia tonsa Dana eggs

The effects of light and short-term temperature elevation on the 48-h egg hatching success (HS) of cold-stored (2 °C) Acartia tonsa Dana (Copepoda: Calanoida) eggs were examined in the present study. The eggs can be stored for up to 7.5 months and maintain their high hatching rate under optimal conditions. Intensively produced eggs from the copepod A. tonsa may be hatched and used as an inoculum for producing copepod nauplii as live feed for fish larvae. The HS for eggs that were directly exposed to LED light declined rapidly after 1 month of storage (from 91 to 25 %), and these eggs did not hatch at all after 3 months of storage. The highest HS found was for eggs stored in complete darkness. The HS for eggs stored in normoxic (≥7 mg DO L^?1) and anoxic (≤0.03 mg DO L^?1) seawater was not affected by short-term temperature transitions from 2 °C up to 9 or 17 °C for a period of 12 or 24 h, when hatched 1 week post-exposure. The global mean HS for eggs stored in normoxic seawater was 85.9 % and significantly lower compared to eggs stored under anoxic conditions after 3 weeks of storage (91.8 %) (P = 0.001; SNK).     

Salinity-induced quiescence in eggs of the calanoid copepod Acartia tonsa (Dana): a simple method for egg storage

We report the effect of salinity and temperature on the viability of stored culture-based subitaneous eggs of the calanoid copepod Acartia tonsa for use of copepods in fish larvae culture. Quiescence induction was recorded at 17 and 25 °C, in salinities from 0 to 30. Quiescence was strongly induced at 0 salinity and partially at 5 in both temperatures. Eggs incubated at 0 salinity for up to 12 days at both temperatures showed a decline in the fraction able to be induced into quiescence by abrupt salinity changes. The hatching success of eggs that were able to enter quiescence stabilized after a 1-day incubation and remained ∼25% viable for 12 days in 17 °C. On the contrary, the 25 °C trial showed a gradual decline in viability until stabilizing ∼10% at day 7 and onwards. Longterm 17 °C incubation for 35 days showed that eggs remained quiescent with a viability of ∼14%. Hence, we recommend salinity storage of A. tonsa subitaneous eggs as a relevant shortterm technique, and a suitable alternative to the recently proposed cold storage of eggs when eggs are to be shipped from the copepod producer to a given fish larvae hatchery.     

The influence of triiodothyronine (T<Subscript>3</Subscript>) on the early development of piracanjuba (<Emphasis Type="Italic">Brycon orbignyanus</Emphasis>)

This paper reports the triiodothyronine’s (T₃) effects on the early growth and survival of piracanjuba (Brycon orbignyanus) produced from fertilized eggs hormone exposed. The study was carried out in two phases. In the first phase, eggs divided in 6 batches were immersed in T₃ solutions: 0.01; 0.05; 0.1; 0.5 ppm; 1 ppm and control (no T₃). After a 15-min immersion, eggs were transferred to incubators where larvae were kept up to 72 h after hatching. Larval weight, length and yolk sac volume were determined every 12 h. Sixty and 72 h after hatching, larvae exposed to 0.5 ppm T₃ were significantly heavier than the others, and those exposed to 1 ppm T₃ showed the lowest weight. The yolk sac absorption was not affected. In the second experimental phase, the resulting fry from the first phase were stocked into 3 boxes per treatment (5 larvae L⁻¹) and fed with plankton, fish larvae and feed prepared in the hatchery (48% CP) in the first 3 days, plankton and feed from the 4th to the 10th day and only feed in the next (last) 5 days. Fry weight, length and specific growth rate were determined at 1, 5, 10 and 15 days. Survival was calculated in the last day. In the 15th day, fry length did not differ among treatments but the weight of the control group was higher. Higher survival in the T₃-treated groups suggested lower predation among fry. The results allowed us to conclude that there was no expressive effect of T₃ on the growth, but it improved the survival of the piracanjuba progeny.     

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.     

Dietary effects on egg production,egg‐hatching rate and female life span of the tropical calanoid copepod Acartia bilobata

Copepods are crucial source of live feeds in the aquaculture industry. In particular, several species of the genus Acartia are considered optimal prey for fish larvae. The species Acartia bilobata has excellent potential for marine larvae culture, as it is easy for mass culture. This study investigated the effects of various algal diets on the egg production and egg‐hatching rate of A. bilobata. The results indicated that the single‐species diet Isochrysis galbana was the most supportive diet for A. bilobata egg production and female life span in all treatments (egg production: 23.85 ± 0.70 eggs female^?1 day^?1 and female life span: 18.00 ± 1.45 days). Nannochloropsis oculata and Tetraselmis chui treatments gave markedly lower egg production and female life span as both single‐species and multiple‐species diets. For the egg hatching‐rate experiment, except for the T. chui treatment, which yielded a considerably lower hatching rate than the other diets, the hatching rate was only slightly affected by the algal diets. These results confirm that A. bilobata, a tropical brackish‐water copepod species, develops rapidly at 28°C and can produce a large number of eggs; therefore, it has considerable potential for larvae culture.