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.     

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.     

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.     

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.     

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.     

Effects of egg disinfection and incubation temperature on early life stages of spotted wolffish

Eggs of spotted wolffish (Anarhichas minor Olafsen)were incubated at constant 4, 6 and 8 °C, and disinfected withglutaric dialdehyde (150 p.p.m. for 5 min) once ortwice a month during two thirds of the incubation period, to prevent growth ofmicroorganisms. Hatching of apparently normal larvae started earlier when eggswere disinfected twice a month compared to once a month at all incubationtemperature regimes. The time to 50% hatch was 900 and 920 day-degrees (16 and16,5 weeks) at 8 °C, 835 and 880 day-degrees (20 and 21 weeks)at 6 °C and 725 and 800 day-degrees (26 and 28,5 weeks) at 4°C, in the egg groups disinfected twice or once a month,respectively. The best survival until hatching was noted when eggs weredisinfected twice a month and incubated at 6 and 8 °C.Survivalwas very low at 4 °C. Prematurely hatched larvae wereregistered in all egg groups disinfected twice a month and the highestfrequencywas noted in the 8 °C groups. The larval weight at normalhatching in the 6 and 8 °C groups was negatively correlatedwith incubation temperature and intervals of disinfection during the incubationperiod, but after 42 days feeding with live feed (unenrichedArtemia) the weights of the larvae were not significantlydifferent. The specific growth rates of the larvae from the eggs incubated at 6°C and 8 °C were 3.0% and 3.2%, respectively.The mean survival of larvae was between 88% and 96% at 42 days post-hatching.Young wolffish originating from the 6 °C incubation groupsshowed lowest mortality.     

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.     

Efficacy of formalin and povidone–iodine disinfection techniques on the eggs of three marine finfish species

Surface disinfection trials were performed on eggs from three marine finfish species: California yellowtail (CYT; Seriola lalandi), white seabass (WSB; Atractoscion nobilis) and California halibut (HA; Paralichthys californicus). All three species were spawned from captive populations maintained at the Hubbs‐SeaWorld Research Institute (HSWRI). Five disinfection treatments were used for each species; Treatment 1 included 100 mg L^?1 of formalin (F100) for 60 min (current HSWRI treatment), Treatment 2 included 1000 mg L^?1 of formalin for 15 min (F1000), Treatment 3 included povidone–iodine of 50 mg L^?1 for 15 min (PI50), Treatment 4 included povidone‐iodine of 100 mg L^?1 for 10 min (PI100) and Treatment 5 involved a control with no chemical treatment (CONT). For each treatment, the per cent egg hatching rate, per cent survival to first feeding and notochord length at the time of hatching to the nearest 0.1 mm were recorded. Bacteria were also cultured from eggs after treatment to determine the effectiveness of each treatment in reducing the bacterial counts (CFU mL^?1). Treatments F100, F1000 and CONT yielded the highest hatch rates for each species (70–80%), whereas treatments PI50 and PI100 yielded the lowest hatch rates (0–2%). There were no significant differences in survival to first feeding or notochord length, which suggests that the disinfection treatments did not have a negative effect on the yolk sac larvae. The PI50 and PI100 treatments had the lowest bacterial colony counts, showing almost zero bacterial growth. The highest bacterial growth occurred in the F100, F1000 and CONT treatments. Based on the results from this study, the F100 treatment provided the best balance of disinfection and larval health for CYT, WSB and HA.     

Progress towards the identification of the sex-determining mechanism of the sole,Solea solea (L.), by the induction of diploid gynogenesis*

Methods were determined for the induction of diploid gynogenesis in the sole, Solea solea (L.), in order to investigate the sex-determining mechanism. The experiments utilized gametes obtained by dissection of fish caught at sea. Activation of eggs with UV-irradiated sole sperm was not feasible but development was initiated by exposure to halibut, Hippoglossus hippoglossus (L.), sperm. These embryos displayed the typical characteristics of haploids and few survived beyond hatching. High levels (> 90%) of diploidy were induced in such eggs by subjecting them to a cold shock (2°C or 4°C) for 1-2 h about 10-15 min after activation at 12°C. The resultant embryos were indistinguishable from those developing from normally fertilized eggs and were considered to be diploid gynogenomes. Fifteen of these were reared to a length of 5-10 cm by which time gonad differentiation had been initiated. Both sexes were represented, indicating that sex in this species is not determined by a simple XX-XY system with female homogamy.     

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.     

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.     

Effect of water hardness on egg hatchability and larval viability of Clarias gariepinus

Studies were conducted to determine the effect of water hardness onClarias gariepinus egg hatchability and larval viability.The fertilized eggs were incubated at 28 °C and with waterhardnesses ranging from 10–700 mg/l CaCO₃. Themean hatching rate fluctuated between 42.31% at hardness of 10mg/land 64.66% at 2000 mg/l. Abnormalities in the larvae were observedbeyond 200 mg/l and increased with increase in water hardness. Thehighest larval survival of 71.05% was recorded at 60 mg/l waterhardness. Based on statistics performed with analysis of variance (ANOVA) andfurther compared with Duncan's multiple range test (p = 0.05), the resultsimplythat very soft water (0–10 mg/l) and very hard water (300mg/l and above) are not suitable for Clariasegg incubation and larval rearing. A water hardness of 30–60mg/l CaCO₃ is recommended for optimal normal hatching,high viability and maximum larval development of Clariasgariepinus.     

四种消毒剂对摇蚊幼虫杀菌效果研究

为筛选四种鱼用消毒剂对摇蚊幼虫(Chironomid larvae)最适消毒条件,在单因素试验基础上,以消毒剂对摇蚊幼虫携带细菌相对灭菌率为考察指标进行正交试验;以消毒后摇蚊幼虫相对存活率、稀有鮈鲫(Gobiocypris rarus)对摇蚊幼虫平均摄食时间、消毒后未杀灭细菌种类为指标,综合评价消毒虫体质量。结果显示:浓度因子对聚维酮碘、新洁尔灭、高锰酸钾的相对灭菌率有极显著影响;时间因子对聚维酮碘的相对灭菌率影响极显著;温度因子对聚维酮碘的相对灭菌率影响极显著,对新洁尔灭影响显著;浓度、时间和温度因子均对戊二醛的相对灭菌率无显著影响;在实验室条件下,根据消毒剂对相对灭菌率的各影响因子主次顺序得到了四种消毒剂消毒摇蚊幼虫的最佳因子参数,其中高锰酸钾:消毒剂浓度60 mg/L,消毒温度28℃,消毒时间1.5 h;聚维酮碘:消毒温度24℃,消毒剂浓度150 mg/L,消毒时间1 h;新洁尔灭:消毒剂浓度3 200 mg/L,消毒温度24℃,消毒时间1 h;戊二醛:消毒温度28℃,消毒时间1.5 h,消毒剂浓度20 000 mg/L。四种消毒剂最佳消毒条件对摇蚊幼虫相对存活率及对稀有鮈鲫摄食效果影响不大;消毒后从不同来源的四份虫体样品共分离出未杀灭的细菌16种,主要隶属于气单胞菌属和不动杆菌属。     

Maximizing lobster, Homarus gammarus (L.), egg and larval viability

The viability of batches of larvae reared to produce lobsters, Homarus gammarus (L.), for stock-supplementation experiments varied widely and rendered the timing and numbers of juveniles available for release unpredictable. Egg lipid reserves, time of hatching, exposure to low salinity during incubation and the influence of feeding regimes on water quality during larval culture were among the factors judged most likely to have affected viability. There was considerable variation in the lipid content of eggs from different females captured in 1989 and 1990. The proportion of lipid in eggs from females caught in 1990 was higher (10-20%) than that in eggs from females caught in 1989 (6-11%). The fatty acid content of eggs declined 72-80% during development although the overall proportions of fatty acids remained similar. Analysis of eggs and larvae from a single brood showed that rapidly developing eggs and the larvae that hatched from them contained greater triacylglyceride lipid reserves (by 49% and 15% respectively) than those which developed more slowly and hatched later. Exposure to reduced salinity (below 29 psu) prolonged development (by 30%), increased losses (to > 50%) and reduced the ratio of n-3:n-6 fatty acids (from 35-48 to 18-29) of eggs being incubated by captive, wild-caught, female lobsters. Larvae hatching from eggs held in reduced salinity (23 psu) survived less well (3% compared with 15%) than those incubated in sea water of 29 psu. The rate and degree to which fouling organisms became attached to larvae was related to feeding regime but not to development rate. A diet of mysids supplemented with mussel produced most fouling, generally poorer growth and a higher incidence of moulting abnormalities than a diet of mysids supplemented with Artemia nauplii. Among larvae fed Artemia, those developing early were significantly larger at instar 4 than late developers (56.4 mg cf. 49.8 mg, P < 0.05), but such differences in weight were not maintained by juveniles during the following 31 days of culture. The ecological and aquacultural implications of the results are discussed.     

Development of wolffish eggs at different temperature regimes

Embryonic development of common wolffish (Anarhichas lupus L.) was studied at constant temperatures 5.0, 7.0, 9.0, 11.0, 13.0 and 15.0°C. Duration of development from egg activation to several morphological stages including 50% hatching was determined. At 5.0–11.0°C, the survival rate of eggs to hatching ranged from 51 to 88% with a tendency to increase at 5.0 and 7.0°C. Morphological anomalies, bacterial contamination and large mortalities were observed in eggs incubated at 13.0 and 15.0°C. The period of hatching lasted from 10 to 50 d in different egg groups. Embryo length and yolk sac volume at identical morphological stages of development showed only slight relation to temperature. At lower temperatures newly hatched larvae were longer and at more advanced stages of ontogeny. Normal numbers of fin rays in larvae (mean values 74 for dorsal fin and 46 for anal fin) were observed at 5.0 and 7.0°C and in most larvae at 9.0°C. At 11.0 and 13.0°C, many rays were absent, with mean values for dorsal fin 60 and 39 respectively and for anal fin 28 and 4 respectively. The approximate upper limit for normal development of fin rays appeared to be 9.0 °C.     

Phenotypic effects of antibiotic‐induced axenity and egg disinfection in early larval European seabass (Dicentrarchus labrax L.)

In the present study, the hypothesis that the difference in axenic conditions in the incubation and rearing environment of European seabass larvae induces size and shape effects on the specimens is tested. This difference is studied between xenic and axenic seabass larvae of DAH (day after hatching) 0, 5, 11 and 15. The axenic rearing protocol involves an egg disinfection with glutaraldehyde after the primary one with iodine in the hatchery, and the hypothesis that this secondary disinfection induces size and shape effects is also tested. In order to accomplish this, three egg and larvae treatments are included: “DA” (disinfected axenic), “DX” (disinfected xenic) and “NX” (nondisinfected xenic). Regarding the effect of antibiotic‐induced axenity, DA larvae exhibited larger bodies than both DX and NX on DAH 5 and 11. They also had a smaller yolk sac than DX at hatching, but consumed it slower. Towards the end of the experiment, DA larvae were thicker, but slightly more curved than DX and NX, which may be an abnormal shape, or a slightly more advanced ontogenetic stage. As far as egg disinfection, it had significant but very moderate shape effects on DAH 5 and 11, and disinfected larvae consumed their yolk sac faster. To the best of our knowledge, this study is one of the first to illustrate the subtle but significant size and shape effects caused by antibiotic‐induced axenity and secondary egg disinfection in the early larval stages, which suggest the existence of bacterial mechanisms that play a phenotypic role.     

Effects of Environmental Factors on Egg Development and Hatching of Pacific Halibut Hippoglossus stenolepis

Eggs of Pacific halibut were incubated under various environmental conditions. Optimum hatching occurred over a temperature range from 6 C to 8 C, whereas temperatures of 3, 10, and 11 C were lethal. Development time from fertilization to 50% hatching varied from 250 h (9 C) to 320 h (6.5 C). Salinity effects on hatching were not as critical as temperature, as long as eggs were floating during the incubation period. Light intensity between 5 and 15 lux did not affect hatching success, but high light intensity (15 lux) and red and blue light (5 lux) produced high levels of larval abnormality. Simulated transport of unfertilized eggs indicated that the eggs can be safely moved and that fertilization rate is acceptable during the first 12 h after collection.     

Incidence of conjoined twins in tilapia after thermal shock induction of polyploidy

Conjoined twins (polyembryonic fish) were observed in batches of blue tilapia Oreochromis aureus (Steindachner) and Nile tilapia O. niloticus (L.) embryos incubated at 28 °C. Heat shock (41.8 °C) treatment of fertilized eggs for 3–4 min applied between 4 min and 27 min after fertilization increased the twinning rate by three or four times over that of unshocked eggs. Two basic morphological variations – ventrally or laterally conjoined twins – were observed. Twins did not survive beyond 21 days after hatching. The proportion of twins in heat‐shocked egg batches of both species combined ranged from 0.32% to 0.40%.     

维生素C对圆斑星鲽早期发育的影响

本研究旨在探究维生素C(V_C)对圆斑星鲽(Verasper variegatus)早期发育中孵化率、出膜率和成活率的影响,对其消化酶和脂蛋白脂酶(Lipoprotein lipase,LPL)及肝脂酶(Hepatic Lipase,HL)的影响。实验用圆斑星鲽为人工受精卵,分为5组,孵化水体V_C浓度分别为0、20、25、30和35 mg/L,分别在成熟卵、受精卵、原肠中期、晶体出现期、内源营养期(2日龄)、混合营养期(6日龄)及外源营养期(9日龄)取样。记录圆斑星鲽的出膜时间、出膜率和成活率,测定对照组和生长指标优越组的消化酶活性和2种酯酶的活性。结果显示,25 mg/L的V_C能显著提高圆斑星鲽的出膜率和成活率,并缩短出膜时间(P0.05)。V_C能促进圆斑星鲽早期发育过程中蛋白质的沉积。外源营养期后,胃蛋白酶活性显著提高(P0.05),淀粉酶活性在混合营养期显著提高(P0.05),脂肪酶和胰蛋白酶活性在内源营养期就显著提高(P0.05)。25 mg/L V_C溶液浸泡使圆斑星鲽胚胎发育过程中的LPL和HL比活力显著升高(P0.05)。合子中的LPL和HL基因可能在原肠期开始表达,合成脂质水解酶分解脂质,为胚胎发育供能。研究表明,圆斑星鲽成熟卵受精后脂肪酶和胰蛋白酶酶优先发挥作用,分解卵黄中的碳水化合物为胚胎发育迅速提供能量。25 mg/L的V_C能显著提高消化酶活性,并促进蛋白质的沉积,有利于孵化成活的V_C浓度可以显著促进圆斑星鲽早期发育的脂质代谢。     

Gynogenesis induction and sex determination in the Eurasian perch,Perca fluviatilis

In the present study, we used meiotic gynogenesis, widely used in studies on sex determination, to confirm female homogamety in Eurasian perch, Perca fluviatilis. Sperm irradiated with UV for 400 s was used to artificially fertilized eggs. The diploid of the resulting embryos was restored by a heat shock (30 °C) applied to the eggs 5 min postfertilization, for 25 min. Fertilization (ranging between 45% and 75%) and survival rates at hatching (ranging between 3.4% and 46.6%) were not significantly different (P>0.05) between the diploid control and gynogenetics. The diploid controls and two batches of gynogenetics contained 100% diploid larvae, whereas two other batches of gynogenetics contained 6.7% and 10.0% triploid larvae. The sex ratios of the diploid controls were not significantly different from 1:1, whereas all gynogenetic families were 100% female. These results confirm female homogamety in Eurasian perch, demonstrated by the use of hormonally mascilinized breeders in a previous study.