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).     

Ultraviolet light and semi‐recirculating systems in artificial incubation of noble crayfish (Astacus astacus) eggs: opportunities and limitations

Development of artificial crayfish egg incubation is a milestone in intensive culture of crayfish as commercially important freshwater animals. This study evaluated experimental treatments combining continuous UV lighting, a non‐chemical antifungal treatment, with an initial formaldehyde bath for noble crayfish (Astacus astacus) eggs incubated in semi‐recirculating systems, which requires less than 1 per cent the amount of water necessary for conventional flow‐through systems. The one‐time administration of a pre‐incubation bath to reduce formaldehyde exposure was ineffective. Ultraviolet irradiation of recirculating water provided poor results (13.5% and 35.2% final survival rates to stage 2 juveniles) and led to deteriorating water quality. An inability of hatchlings to successfully moult and the occurrence of limb deformities was observed in UV‐treated groups, and juvenile mortality was found across all experimental treatments.     

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.     

The use of formaldehyde for the disinfection of maternally incubated eggs of noble crayfish (<Emphasis Type="Italic">Astacus astacus</Emphasis>)

The high mortality of maternally incubated eggs represents a serious problem that prevents the development of noble crayfish (Astacus astacus) farming in Italy. In this experiment, formaldehyde bath technique has been tested with maternally incubating females. The survival rate of maternally incubated eggs, exposed to progressively reduced concentrations of formaldehyde (4000, 3000, 2000, 1000, 1500 and 500 mg l^?1), has been compared with that of untreated controls for two consecutive years (2010 and 2011). The formaldehyde treatments were administered as disinfection baths, for a period of 15 min, twice a week. A concentration of formaldehyde of 500 mg 1^?1, or greater, was found to be effective in controlling egg mortality, without apparent harming the female broods.     

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%.     

基于循环水养殖系统的长鳍吻鮈亲鱼培育、催产和孵化技术初探

2012—2015年,采用室内循环水养殖系统开展了长鳍吻鮈(Rhinogobio ventralis)亲鱼培育、人工催产和孵化技术研究。结果显示:3年间长鳍吻鮈均能在循环水养殖系统中培育成熟,2014年5月—2015年4月间培育存活率和成熟率最高,分别为85.3%~100%和77.3%~100%;对培育成熟的亲鱼进行了4种不同外源激素(PG、HCG、LRH-A2、LRH-A2+DOM)催产实验,结果为LRH-A26μg/kg+DOM 5 mg/kg合剂催产率最高,达88.3%;14、16、18、20和22℃5个温度组的受精卵孵化实验结果显示,孵化时间(H)与温度(T)呈显著负相关关系(H=0.573T2-30.393T+418.178,R2=0.982,P0.01),18℃和20℃组的孵化率分别为54.27%和56.07%,显著高于其他各组,表明长鳍吻受精卵适宜孵化温度范围为18~20℃。     

Artificial incubation of Japanese crayfish (Cambaroides japonicus) eggs by using a simple, easy method with a microplate

We developed a simple, easy method with a microplate to artificially incubate Japanese crayfish (Cambaroides japonicus) eggs for their cultivation. We prepared 6-, 12-, 24-, 48-, and 96-well microplates containing sterile water heated to 5, 10, 15, and 20 °C. Fourteen experimental groups for each water temperature were prepared for each of different water volumes (0.125–10 ml) in each well. One embryonic egg was placed in each well. Experiments were also conducted with water collected from the lake where the eggs were harvested from and held at 15 °C. In the microplates with sterile water, high proportions of eggs hatched (60–100%) at 15 °C in all volumes of water, although the proportions of hatching were low (0–20%) at 5, 10, and 20 °C. All eggs died in the experiments that used lake water. We conclude that the 96-well would be the most effective size to hatch crayfish eggs in, because of its convenience. This method using a microplate is simpler and easier compared with methods of previous studies to artificially incubate crayfish eggs, and therefore it might be useful to incubate eggs of other freshwater crayfish species.     

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.     

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.     

Small-scale fry production systems for Nile tilapia, Oreochromis niloticus (L.)

Abstract Experiments were conducted to evaluate and compare the production, quality and survival of eggs and the subsequent growth and survival of fry in two small-scale production systems: natural incubation and egg collection followed by artificial incubation. Artificial incubation produced a significantly greater number and biomass of fry per unit weight of female broodstock (P < 0·05). This was due to the significantly decreased spawning interval in females from which eggs were collected (mean = 23· days) compared to naturally incubating females (mean = 36·8 days). It is also hypothesized that survival during artificial incubation (85%) is greater than in natural incubation. Cost-benefit analysis demonstrated a threefold increase in total net returns for artificial incubation.     

Prevention of Saprolegniasis in rainbow trout (Oncorhynchus mykiss) eggs using oregano (Origanum onites) and laurel (Laurus nobilis) essential oils

The present study investigated the antifungal effects of essential oils of oregano (Origanum onites) and laurel (Laurus nobilis) on Saprolegniasis, a disease that occurs in rainbow trout eggs during the incubation period. Oregano and laurel were ground after drying, and essential oils were obtained by water distillation method using a Clevenger apparatus. The essential oils were added to potato dextrose agar (PDA) at the rates of 1–1000 ppm, and the minimum inhibitory concentration (MIC) was determined as 250 ppm whereas the minimum lethal concentration (MLC) was determined to be 500 ppm for both plants. In the in vivo trials, fertilized eggs were treated with predetermined doses either by bathing during water hardening and incubation period or only during incubation period, and death rates were monitored during embryological development. The best larvae hatching rate was determined in 500 ppm oregano and 500 ppm laurel groups treated during water hardening plus daily as 82.11% and 79.87%, respectively. According to the results, it was determined that oregano and laurel essential oils exhibited better results in all doses compared with the negative control group, and 500 ppm dose had a better effect than the positive control group treated with formalin.     

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.     

Disinfection of striped trumpeter (Latris lineata) eggs with glutaraldehyde

This study assessed the effect of immersing striped trumpeter eggs in 0 (control), 200, 400, 800, 1600 or 3200 ppm glutaraldehyde for 10 minutes, two days before hatching. High concentrations of glutaraldehyde (1600 and 3200 ppm) resulted in no eggs hatching and only 1% of eggs hatched after treatment with 800 ppm glutaraldehyde. Hatching success of eggs treated with 0, 200 or 400 ppm glutaraldehyde did not differ (77 ± 6%, n = 3). However, only 2% of larvae from the control treatment survived to day 5 post-hatching, compared to 45 and 69%, respectively of the larvae from the 200 and 400 ppm glutaraldehyde treatment. By day 9 post-hatching, larvae from the 400 ppm glutaraldehyde treatment had significantly higher survival (59%) than larvae from the 200 ppm glutaraldehyde treatment (28%). Thiosulphate citrate bile salt sucrose agar (TCBS) medium confirmed the presence of bacteria within the seawater medium, on all control eggs and on 83% of eggs disinfected with 200 ppm glutaraldehyde, but no bacterial colonies formed on eggs treated with 400, 800, 1600 or 3200 ppm glutaraldehyde. This study found highest survival of striped trumpeter larvae from eggs disinfected with 400 ppm glutaraldehyde and suggests that increased survival was a result of reduced bacterial loading.     

Effect of the dusk photoperiod change from light to dark on the incubation period of eggs of the spotted rose snapper, Lutjanus guttatus (Steindachner)

Spotted rose snapper, Lutjanus guttatus (Steindachner), eggs were incubated under different photoperiods to examine the effect of photoperiod on incubation. The eggs from two fish were incubated under five artificial photoperiods: constant dark (D), constant light (L) from 06:00 hours and 6, 10 and 14 h of light from 06:00 hours. The eggs from seven other fish were incubated under a natural photoperiod. Different spawning times (21:00 – 01:00 hours) and different photoperiods combined to give the start of the dusk photoperiod change after 11–23 h of incubation. Constant light or applying the dusk photoperiod change after ≥20 h of incubation appeared to extend the hatching period. The mean hatching period for groups of eggs incubated in darkness or that received the dusk photoperiod change after ≤19 h of incubation (n=8 different groups) was 2 h 15±10 min, which was significantly lower (P<0.05) than the mean hatching period of 4 h±37 min for groups that did not receive the dusk photoperiod change or that received the dusk photoperiod change after ≥20 h of incubation (n=9 groups). However, despite these differences, the majority of the eggs hatched during a 2–3 h period from 17 to 20 h of incubation, and a sigmoid regression (r²=0.9) explained the relationship between percentage hatch and hours of incubation for all photoperiod groups.     

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.     

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.     

Rapid de-adhesion of northern pike <Emphasis Type="Italic">Esox lucius</Emphasis> eggs using sodium hypochlorite

The elimination of egg stickiness is required for effective artificial reproduction of northern pike, but until now, available methods have required at least 40 min. Sodium hypochlorite was tested under laboratory conditions, and exposure to aqueous concentrations of 0.025–0.05% for 40 s effectively eliminated stickiness without adverse effects. Fertilization and hatching rates in laboratory trials were similar to those observed in eggs treated with traditional methods using clay or milk for 40 or 60 min, respectively, as well as those without treatment. Testing using conventional hatchery incubation techniques did not reveal differences in fertilization rates, while the number of hatched larva was significantly higher in eggs treated with sodium hypochlorite vs. clay. Eggs treated with sodium hypochlorite retained transparency, which facilitated monitoring of embryo development.     

红螯螯虾的室内人工育苗

于1996-1998年,在浙北地区,对澳大利亚引进的红螯螯是进行亲虾培育、人工越冬,怀卵孵化,室内人工育苗技术的研究。研究表明,在浙北地区,2.8-4.9g的幼虾经5个月左右的饲养可以达到性成熟并部分怀卵,利用电厂余热水水泥池人工越冬的成活率可达70%以上。越冬后亲是在水温20℃以上即开始交配怀卵,怀卵盛期4-6月。红螯螯虾一年可产卵4次,但只有第一、二次怀卵可用于育苗生产,个体一次怀卵量较少,一般为400-500粒,但群体怀卵比率较高。试验还表明,红螯螯虾出膜幼体即呈幼虾状,需依附母体7-10d后才营独立生活并开口摄食外源性饵料,所研制的幼虾Ⅰ号饲料为红螯螯虾室内人工育苗较好的开口饲料。室内人工育苗成活率可达60%以上。     

The Toxicity of Hydrogen Peroxide to Rainbow Trout Oncorhynchus mykiss and Cutthroat Trout Oncorhynchus clarki Fry and Fingerlings

Fungal and parasitic infections of fish can significantly impact the survival of cultured fish. Formalin is currently used to control such infections; however, concern has arisen over its safety to users and to the environment. Hydrogen peroxide has been designated as a low priority fungicidal drug by the United States Food and Drug Administration (FDA), yet, little information is available on treatment concentrations or its toxicity to trout. Rainbow trout Oncorhynchus mykiss and cutthroat trout Oncorhynchus clarki fry and Angerlings were exposed to hydrogen peroxide concentrations of 0, 70, 170, 280, 420 and 540 ppm for 30, 60 or 120 rnin at 15 C to determine the chemical's toxicity. Rainbow trout fry and fingerlings experienced elevated mortalities (>20%) during treatments using 420 and 540 ppm for 30 min; 280, 420 and 540 ppm for 60 min; and >170 ppm for 120 min. Cutthroat trout fry experienced elevated mortalities (>23%) during treatments using 540 ppm for 30 min; 420 and 540 ppm for 60 min; and >170 ppm for 120 min. Cutthroat trout fingerlings experienced elevated mortalities (>60%) during treatments using 540 ppm for 60 min and >280 ppm for 120 min. No control mortalities were encountered for both life stages of either species. The lethal concentrations (LC₅₀) of both age classes and species for each of the three durations ranged from 514–636 ppm for 30 min treatments, 322–506 ppm for 60 min treatments, and 189–280 for 120 min treatments. Mortalities for all four toxicity tests which occurred during a 96-h post-treatment period were centered around the following treatments: 30 min, 540 ppm; 60 min, 280–540 ppm; 120 min, 170 ppm. Tissue damage to gills was found only among fish that did not survive the initial chemical exposure. Test concentrations proved to be relatively stable during a 24-h period, retaining better than 85% of their original strength for all five dilutions. At a water temperature of 15 C concentrations should not exceed 280 ppm for a 30-min treatment.     

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.