Breakpoints for disc diffusion susceptibility testing of bacteria associated with fish diseases: A review of current practice

A survey of the methods being employed to determine antimicrobial susceptibility of bacteria associated with aquaculture was performed on behalf of the Permanent Advisory Network for Diseases in Aquaculture. Thirty-two laboratories in 18 countries responded and 25 reported the breakpoints they used for disc diffusion assays applied to Group 1, non-fastidious organisms isolated from finfish. A total of 117 breakpoints were reported for assays in which the disc contents were those specified by the current standard protocols. Data on the source of these breakpoints and the confidence the laboratories had in them are presented. Overall there was a considerable variation in the breakpoints employed by different laboratories and this variation is discussed in terms of the inter-laboratory precision that can be expected from the application of disc diffusion protocols. This paper discusses the possible clinical significance of the variations in the breakpoints and, where there are available data, the extent to which those in use are consistent with breakpoints suggested by other approaches.The data presented in this paper represent a starting point for the movement towards harmonising breakpoints used in association with the standard disc diffusion protocols that have been proposed for susceptibility testing of bacteria associated with fish diseases.     

Influence of disc content on zone sizes obtained in standard disc diffusion antimicrobial susceptibility tests

The influence of disc content on the zone sizes produced was investigated using standard disc diffusion antimicrobial agent susceptibility test protocols. Replicate measurements were made of the zones of inhibition produced by discs containing 2-64 μg of oxytetracycline and oxolinic acid against Escherichia coli, Aeromonas salmonicida and Vibrio parahaemolyticus. Five replicate data sets were obtained for each of the six organism-agent combinations. Analysis of these 30 data sets demonstrated that they all showed a very high correlation (mean r² = 0.98 ± 0.009) with the predictions of best-fit equations specifying a linear relationship between log₁₀ of the disc content (μg) and the resultant zone sizes (mm). The mean value of the slopes of the best-fit straight-line equations was 9.8 ± 1.8. The overall similarity of these values for the slopes suggested that the quantitative relationship between disc content and zone size was, at least to a first approximation, independent of the organism or the agent involved in the test.     

Effect of incubation temperature and time on the precision of data generated by antibiotic disc diffusion assays

The influence on the precision of disc diffusion data of the conditions under which the tests were performed was examined by analysing multilaboratory data sets generated after incubation at 35 °C for 18 h, at 28 °C for 24 h and 22 °C for 24 h and 48 h. Analyses of these data sets demonstrated that precision was significantly and progressively decreased as the test temperature was reduced from 35 to 22 °C. Analysis of the data obtained at 22 °C also showed the precision was inversely related to the time of incubation. Temperature and time related decreases in precision were not related to differences in the mean zone sizes of the data sets obtained under these test conditions. Analysis of the zone data obtained at 28 and 22 °C as single laboratory sets demonstrated that reductions of incubation temperature resulted in significant increases in both intralaboratory and interlaboratory variation. Increases in incubation time at 22 °C were, however, associated with statistically significant increases in interlaboratory variation but not with any significant increase in intralaboratory variation. The significance of these observations for the establishment of the acceptable limits of precision of data sets that can be used for the setting of valid epidemiological cut‐off values is discussed.     

An apparatus for long term biological experiments in running water at temperatures close to freezing

An open aquarium system in which water temperatures can be manipulated and controlled close to freezing point has been constructed. Technical details and the results of a working trial lasting six weeks are reported.

The equipment was constructed in order to be able to simulate river water temperatures, and changes in these due to hydroelectrical development in Arctic regions. The incubation of Atlantic salmon eggs was of particular interest. Other applications include growth studies and nutritional physiology of aquatic organisms at low temperatures. The system can be used with both freshwater and seawater.     

Effect of dietary protein and lipid source on the growth, survival, condition indices, and body composition of marron, Cherax tenuimanus (Smith)

A trial was conducted in 12 purpose-built, commercial, drainable, earthen ponds to evaluate the effect of fish and plant protein and lipid source on the growth, condition indices, and body composition of marron (Cherax tenuimanus). Juvenile marron (1.3±0.28 S.E. g) at the stocking densities of three per square meter were fed for a period of 1 year with four different formulated isoenergetic practical diets (D₁, D₂, D₃, and D₄). Three of the test diets (D₁, D₂, and D₃) were isonitrogenous whereas the fourth test diet (D₄) was protein-free. Protein and lipid sources in D₁ were from Lupin (Lupinus albus) whereas protein and lipid sources in D₂ and D₃ were from fish meal. Fish oil (3.5%) was added to D₁, D₃, and D₄ whereas sunflower oil was added to D₂ and D₄ in order to make them isoenergetic. The four test diets were randomly allocated to three replicate ponds.

The lack of protein in D₄ did not significantly influence (P>0.05) the mean final weight and specific growth rate of marron. Survival was low in all ponds (13.82–34.66%) but feeding with D₄ resulted in a significantly (P<0.05) higher survival than marron fed with D₁ and D₂. Feeding a diet containing a combination of fish protein and fish oil (D₃) resulted in significantly higher (P<0.05) wet tail muscles-to-body weight ratio than was observed with other diets. Tail muscles protein level of all marron was significantly lower (P<0.05) at the end of the trial than at the beginning. EPA and DHA in hepatopancreas and tail muscles of marron were affected by the four test diets. The inclusion of plant protein in formulated diets had a negative impact on the pond environment due to significantly higher unionised ammonia levels that resulted in lower survival. Juvenile marron fed with a plant protein diet had significantly lower protein levels in their hepatopancreas compared to those fed with diets containing animal protein. Feeding marron with lupin protein source (D₁) and plant oil (D₂) for 1 year did not alter the lipid content of their hepatopancreas. Four test diets had no influence on the fat content of marron hepatopancreas; however, these test diets significantly reduced the protein content of the tail muscles of marron.

Environmental variables, particularly temperature, nitrogen metabolites, and the natural productivity of the ecosystem, greatly influenced the nutritional requirements of the juvenile marron under culture.     

Accumulation of aluminum on atlantic salmon eggs during the incubation period

Groups of rainbow trout Onchorhyncus mykiss eggs were incubated in duplicate trays with PVC screens and the original aluminum screens. During the incubation period, aluminum bound to sediments was deposited on the eggs and in the whole hatching system.

The pH of the inlet water to the hatching system with PVC screen was experimentally lowered by adding sulphuric acid. Buffering of the outlet water was observed at pH lower than 5, and at pH 4·5 and lower, an increased aluminum concentration in the outlet water was recorded.

These experiments show that aluminum accumulated on eggs and in the hatching system during periods with neutral water represents a serious threat to the survival of eggs and fry of salmonids if pH drops below 5·0, even for a few hours. In sensitive lakes and rivers, pH drops below 5·0 are common during heavy rain or during the snow melting period.     

Epidemiological cut-off values for disc diffusion data generated by standard test protocols from <Emphasis Type="Italic">Edwardsiella tarda</Emphasis> and <Emphasis Type="Italic">Vibrio harveyi</Emphasis>

The susceptibility of 52 isolates of Edwardsiella tarda and 54 isolates of Vibrio harveyi to 12 antibiotics was established using internationally standardised disc diffusion protocols. Normalised resistance interpretation was employed to generate statistically valid epidemiological cut-off values for these susceptibility data. For three of these 24 species/antibiotic data sets, there were insufficient fully sensitive strains to allow analysis and for an additional two there were sufficient fully susceptible isolates to allow only a provisional estimate to be made. The data sets for a further two species/antibiotic combinations were considered too diverse to allow the calculation of a valid cut-off value. Valid epidemiological cut-off values were, therefore, calculated for 17 species/antibiotic combinations. It is argued that these cut-off values will greatly facilitate the performance of studies designed to monitor the consequence of antibiotic use in aquaculture. They will also facilitate the prudent and rational choice of antibiotics to be applied therapeutically in commercial farming of aquatic animals.     

International study on Artemia: XIX. Hatching data for ten commercial sources of brine shrimp cysts and re-evaluation of the “hatching efficiency” concept

A comparative hatching study has been carried out with Reference Artemia Cysts as well as cysts from Argentina, Australia, Brazil, Canada, France, Italy, the People's Republic of China, the Philippines and the U.S.A. Hatching rate, percentage and efficiency vary considerably from one cyst source to another. These hatching criteria are, however, not strainspecific since significant variation is found among cyst batches from the same geographical origin.

The limitations of the “hatching efficiency” concept are discussed and a new criterion “hatching output”, i.e. the biomass of nauplii expressed in mg dry weight produced per gram cyst product, is proposed for evaluation of the hatching quality of Artemia cyst brands.

The hatching quality of Reference Cysts and cysts from Canada and Argentina can be significantly improved by incubation of the cysts at low salinity.

For almost all commercial sources the use of decapsulated cysts results in a significant increase of the hatching output.     

尿囊素对印度产紫菜Pyropia chauhanii单孢子放散和体细胞分化的影响

为探讨尿囊素对印度产紫菜Pyropia chauhanii单孢子形成与放散的影响,选用能释放单孢子的野生型品系(PC-WT)与不能释放单孢子的诱变品系(PC-Y1)为实验材料,用含不同浓度尿囊素的培养液分别培养来自叶状体梢、中和基部的圆盘体。结果显示,处理后6 d,不同部位的叶状体圆盘体的单孢子放散量次序为中部梢部基部;随着尿囊素浓度的增加,PC-WT品系叶状体圆盘体的单孢子放散总量呈先升后降的趋势,其中10mmol/L是促进单孢子放散的最适浓度;但尿囊素并不能使原本不放散单孢子的PC-Y1品系释放单孢子。用含20 mmol/L尿囊素的培养液培养PC-Y1品系的叶状体圆盘体,8 d后再用酶解法将其体细胞单离出来经体外培养后发现,经尿囊素处理的单离细胞发育成丝状体的百分率为66.1%,而不含尿囊素的对照组仅为15.2%,说明尿囊素对紫菜叶状体的体细胞向生殖细胞分化具一定的促进作用。     

Analysis of sediment transport modeling using computational fluid dynamics (CFD) for aquaculture raceways

A simulation model to analyze the water flow and sediment transport in aquaculture raceways was developed using a computational fluid dynamics (CFD) software package. The simulation was used to evaluate the efficiency of solids settling in the quiescent zone of existing trout raceways. This efficiency was based on the percentage of solids removed, which corresponds to the percentage of solids introduced into the raceway that settle in it, with settling taking place primarily in the quiescent zone.

The raceway selected for model validation was a rectangular concrete raceway 30.0 m long, 3.0 m wide, 0.9 m deep, with a slope of 0.01. The raceway included a quiescent zone of approximately 5.3 m in length, which was separated from the rearing area by a screen. The water flow rate through the raceway was approximately 0.058 m³/s. Velocity measurements were recorded at 230 stations along the raceway using an acoustic Doppler velocimeter, for comparison with the results obtained from the simulations.

For the purpose of simulating sediment transport, six groups of particles were used to account for the total suspended solids. The sizes of the particles selected were based on an experimentally determined distribution for solids from a similar raceway, and were 692, 532, 350, 204, 61, and 35 μm for Groups 1–6, respectively. The particle density for each size was assumed to be 1150 kg/m³. Values of the percentage of solids removed for the different particle sizes were 100.0% for the largest particles, and 54.7, 0.9, and 0.1% for the three smallest particles, respectively. This methodology of analyzing the raceway sediment transport in terms of its percentage of solids removed based on CFD simulations can also be used to examine raceway design alternatives for improving the particle removal efficiency.     

抗生素处理对坛紫菜叶状体生长和藻际微生物的影响

设计了氨苄青霉素、硫酸庆大霉素和卡那霉素3种抗生素的不同组合处理坛紫菜叶状体,旨在筛选出坛紫菜叶状体藻际微生物的最优去除方法;同时,探究紫菜叶状体生长与微生物群落变化之间的关系。结果显示,低浓度(10μg/mL)的硫酸庆大霉素或高浓度(800μg/mL)的卡那霉素处理,对坛紫菜叶状体生长均具一定的抑制作用,而不同浓度的氨苄青霉素处理均促进了叶状体生长。500μg/mL的氨苄青霉素、500μg/mL的硫酸庆大霉素与300μg/mL的卡那霉素3种抗生素混合处理叶状体24 h,不会抑制叶状体的生长;同时,对藻际微生物有较强的抑制性。不同的坛紫菜品系,不同的叶状体日龄和培养密度,以及在不同的海水盐度和不同浓度的营养盐中培养,抗生素处理组圆盘体的生长均比对照组快。通过比较抗生素处理组与对照组分离的菌株后发现,抗生素处理组有两株菌落形态和菌株形态与对照组显著不同的特有菌株。研究表明,在坛紫菜叶状体的生长过程中,某些对叶状体生长起阻碍作用的细菌如果大量繁殖,会抑制对叶状体生长有益的细菌繁殖,当用抗生素抑制了阻碍生长的细菌,就会使益生菌成为优势菌,促进叶状体的生长,益生菌对上述抗生素的处理不太敏感,但由于培养条件的改变,抗生素对阻碍生长的细菌的抑制作用会减弱,使得它们大量繁殖,从而抑制叶状体的生长。     

Early-weaning diets for gilthead sea bream (Sparus aurata L.) and their potential use in Hellenic marine fish hatcheries

The purpose of this experiment has been to evaluate the suitability of commercially available early-weaning microdiets (MDs) for the production of sea bream early juveniles and in comparison with late-weaning protocols already in use by Hellenic marine fish hatcheries. Four sea bream experimental groups were allocated in rearing tanks of a commercial Hellenic marine fish hatchery. Each group represented a different protocol (A, B, CA, and CB) based on the combination of two different early-weaning MDs (A and B) and a late-weaning diet (C). In addition, the late-weaning protocols have received Artemia instar II only and not Artemia instar I. In protocol A, Artemia instar I first feeding and the early-weaning diet A were administered at 17 days post-hatch (17 dph). In protocol B, Artemia instar I first feeding started at 15 dph and the early-weaning diet B was administered at 18 dph. In the C protocols, Artemia instar II first feeding started at 20 dph and the early-weaning diets (A or B) were administered at 25 dph. All protocols have received the late-weaning diet C only after the 50th dph. The experiment lasted for 65 days. By the end of the experiment, early-weaning protocol A and late-weaning protocol CB had similar wet weights, but still lower to the wet weights recorded for the late-weaning protocol CA. These results cannot be explained solely by the nutritional profile of each weaning diet. The larval fatty acid profile of each protocol and at various time intervals reveals the importance that the succession of Artemia, rotifers, and MDs has for each protocol and not just the nutritional profile of the weaning diets, per se.     

Influence of temperature on protein utilization in juvenile European seabass (Dicentrarchus labrax)

The aim of this trial was to study the utilization of dietary protein by seabass juveniles with 5.5 g mean body weight, at two water temperatures: 18°C and 25°C. For that purpose, the fish were fed for 12 weeks, four isoenergetic diets with different protein levels (36, 42, 48, and 56%). At the end of the trial, growth rate and feed utilization were significantly better at the higher water temperature. Within each temperature, specific growth rate and feed efficiency were significantly higher with the 48 and 56% protein diets than with the other diets. At 25°C, feed efficiency was also significantly better with the 56% than with the 48% protein diet. N retention (g kg average body weight⁻¹ day⁻¹) was higher at 25°C than at 18°C but, as a % N intake the inverse was true. Although at 25°C N retention (% N intake) was not different among groups, retention in g kg ABW⁻¹ day⁻¹ was significantly higher with the 56% protein diet than with 36 and 42% protein diets. On the contrary, at 18°C N retention (g kg ABW⁻¹ day⁻¹) was similar among groups while as a percentage of N intake it was inversely related to the dietary protein level. Regarding energy utilization, at each temperature, there were no differences among dietary treatments on energy retention (g kg ABW⁻¹ day⁻¹). As a % of energy intake, energy retention significantly increased with the increase of dietary protein level at 25°C, while at 18°C, there were no significant differences among groups. Within each temperature, at the end of the trial, there were no differences among groups in proximate composition of whole fish. Apparent digestibility coefficients of dry matter, protein and energy significantly improved with the increase of water temperature but, within each temperature, there were no significant differences among groups. The results of this study indicate that, regardless of water temperature, the dietary protein requirement for growth seems to be satisfied with a diet containing 48% protein. Growth and feed efficiency were significantly higher at the higher temperature, however, protein utilization was more efficient at the lower temperature.     

Marine shrimp aquaculture: a novel waste treatment system

Ecuadorian Penaeus vannamei were cultured in dirt ponds (each of approximately 163 m²) at four different stocking densities, i.e. 5 shrimp m⁻², 10 shrimp m⁻², 15 shrimp m⁻² and 20 shrimp m⁻². Experiments were carried out over three different periods during the year. Each experiment lasted for 11–14 weeks. No commercial feed was given to the shrimp. The only input to the ponds was about 30 kg of cattle manure per pond per week. Chemical composition of the cattle manure was analyzed. Water quality parameters such as temperature, pH, DO and turbidity were recorded twice daily for each experiment; nutrients (nitrite, nitrate, ammonium and phosphate), water ATP, sediment ATP, H₂S and chlorophyll were measured twice weekly for each experiment. Shrimp were sampled either weekly or bi-weekly for body weight measurements.

The results showed a negative correlation between stocking density and growth. Weekly growth ranged from 0·44 to 1·58 g week⁻¹. Survival was over 50% in all treatments and averaged at 70·8%. Under these stocking densities, shrimp production ranged from 4·4 to 18·8 kg ha⁻¹ day⁻¹. The stocking density of 15 shrimps m⁻² provides better production than the other stocking densities.

Water quality data did not relate to any shrimp growth. Water nutrient levels in pond discharge water were less than or equal to the nutrients in the incoming water in spite of the weekly addition of cattle manure and did not increase with the addition of cattle manure. No coliform bacteria were detected in any pond water samples through the study period. This indicates digestion of cattle manure in marine shrimp ponds would not pollute the environment with high concentrations of dissolved nutrients.

Thus, a marine shrimp pond can be considered a dissolved nutrient marine treatment plant converting unwanted cattle manure (1841 kg cattle manure ha⁻¹ week⁻¹ in this study) into a valuable commodity — shrimp.     

Bacterial degradation of antibiotic residues in marine fish farm sediments of Uranouchi Bay and phylogenetic analysis of antibiotic-degrading bacteria using 16S rDNA sequences

ABSTRACT:   Antibiotic residues in marine sediments of fish farms negatively influence microbial ecologic systems. The microbial degradation of antibiotic residues was experimentally examined in the marine sediments of Uranouchi Bay, to which one of five antibiotics was added. After incubation reducing physical factors, ampicillin, doxycycline, oxytetracycline, and thiamphenicol were significantly degraded, while josamycin maintained most of the initial amounts. The isolates resistant to ampicillin, josamycin, oxytetracycline, or thiamphenicol degraded each antibiotic in wide ranges of degrees, whereas the isolates degrading doxycycline were not obtained. Microbial degradation may contribute to the disappearance of ampicillin, doxycycline, oxytetracycline, and thiamphenicol in the fish farm. In contrast, the disappearance of josamycin would depend on physical factors, but the bacteria degrading josamycin at least exist in the marine sediments. Phylogenetic analysis using 16S rDNA sequences demonstrated that the antibiotic-resistant isolates formed several clusters in the Gram-positive bacterial group, the Flavobacterium–Cytophaga–Bacteroides group, and the proteobacteria subdivisions. The antibiotic-resistant bacterial population would be composed of various species including ubiquitous coastal bacterial groups. Several species of antibiotic resistant bacteria show antibiotic degradation activities, and appear to contribute to the disappearance of antibiotics in Uranouchi Bay.     

Influence of feeding ratio and size on susceptibility to microsporidial gill disease caused by Loma salmonae in rainbow trout, Oncorhynchus mykiss (Walbaum)

Two trials were designed to quantify the effect of feeding ratio and fish size on the cohabitation transmission of Loma salmonae, the causative agent of microsporidial gill disease (MGD) in salmonids, Oncorhynchus spp. To evaluate the effect of feeding ratio on disease onset, groups of 45 rainbow trout, O. mykiss (Walbaum) (RBT), were fed daily at 1% (low), 2% (medium) or 4% (high) of the fish biomass in the tank. There were three tanks at each feeding level: two tanks were exposed to the pathogen and one was a control. For the second objective, 300 RBT were separated into seven tanks so that the weight classifications were small (17-23 g), medium (32-38 g) and large (57-63 g). Each size class was done in duplicate with one control tank containing medium-sized fish. Separately for each trial, on day 0 post-exposure (PE) five highly infectious RBT were added to each tank (not including the control tanks) to begin the cohabitation exposure period. Beginning on day 21 PE and continuing biweekly until days 70 and 77 PE for the feeding and fish size trials, respectively, each fish was evaluated for visible branchial xenomas to determine disease onset time. Using survival analysis, the survival curves for the low, medium and high feedings were not significantly different from each other. However, there were significant differences amongst the small, medium and large weight classes in the size trial. The median numbers of days to the development of branchial xenomas was 31, 38 and 42 for small, medium and large size fish, respectively. On any given day, a medium or large sized fish had a hazard ratio for developing branchial xenomas of 0.66 and 0.63, respectively, compared with a small fish. In addition to host species and host strain differences, fish size is now considered a host risk factor for the development of MGD.     

Observation on the spawning of Sardina and Sardinella off the south Moroccan Atlantic coast (21–26°N)

This paper provides information on the early life stages of the Moroccan Atlantic sardine, Sardina pilchardus, and two species of Sardinella: Sardinella aurita and S. maderensis, between Cape Blanc (21°N) and Cape Boujdor (26°N), dealing with the spawning grounds, the nursery areas and the optimal spawning temperature and salinity range.

The spawning areas for Sardina are located North of Dakhla (24°30′–25°30′N) and near Cintra Bay (23°N). The larval development area is described together with seasonal and annual characteristics. The nurseries of Sardina are near Dakhla and to the south between 21 and 22°N but precise locations vary with season. The maximum occurrence of eggs was during winter, and was at temperatures from 16 to 18 °C in winter and between 18 and 18.5 °C in summer.

The main spawning area of Sardinella species is between Cape Blanc and Cintra Bay (21–23°N) with a maximum occurrence of eggs and larvae in July. For Sardinella off the southern region of Morocco, the optimal temperature interval for spawning is between 18 and 21.14 °C. No conclusions can be drawn on the relationship between spawning and water salinity.

The area north of Cintra Bay has a broad, but shallow continental shelf. This topography leads to decreasing dispersion effects and the zone constitutes a favorable area for larval retention and development.     

Enrichment of Artemia nauplii in essential fatty acids with different types of liposomes and their use in the rearing of gilthead sea bream (Sparus aurata) larvae

The present study aimed to optimize the enrichment protocols of Artemia nauplii with highly unsaturated fatty acid liposomes. Three different types of liposomes formulated with krill phospholipid extract were tested, using a commercial emulsion as the control treatment. The incubation time of the nauplii in the enrichment medium was chosen as the first variable studied. Total lipid, lipid class and fatty acid profiles were analyzed in enriched nauplii after 18 and 21 h incubation in the enrichment media. A third sampling point at 24 h, where nauplii had been kept in clean seawater at 28 °C for 3 h, was established to emulate potential changes in the nutritional value of the nauplii after some hours in the larval rearing tank. A second objective of the study was the establishment of a suitable liposome-based diet for the rearing of gilthead sea bream larvae, as a model of marine fish.Differences in the enrichment efficiencies through time were found not only between liposome and emulsion based diets, but also among the different types of liposomes. Whereas nauplii enriched with multilamellar vesicles and unilamellar liposomes prepared by extrusion had maximum enrichment performance at 18 h of incubation, nauplii enriched with unilamellar liposomes prepared by the detergent solubilization method followed the enrichment pattern of the control emulsion, with an optimal incubation period closer to 21 h. Analyses of the diets of the larval nutrition trial seemed to confirm the results obtained in the first experiment, although changes in certain variables occurred among the enrichments carried out during the larval feeding trial. Biochemical characterization of body lipids of the larvae indicated differences in total lipid and essential fatty acid content, although no significant differences were found in growth and survival of the larvae. Thiobarbituric acid reactive substances (TBARS), used to determine the peroxide content of the larvae, indicated a significant higher content of these compounds in larvae fed emulsion enriched nauplii compared to larvae fed liposome enriched prey.     

In vitro hydroxylation of vitamin D3 and 25-hydroxy vitamin D3 in tissues of Atlantic salmon Salmo salar, Atlantic mackerel Scomber scombrus, Atlantic halibut Hippoglossus hippoglossus and Atlantic cod Gadus morhua

The in vitro metabolism of ¹⁴CD₃ and ³H25OHD₃ was investigated in different tissues from Atlantic salmon Salmo salar , Atlantic mackerel Scomber scombrus , Atlantic halibut Hippoglossus hippoglossus and Atlantic cod Gadus morhua . The tissues analysed were liver, kidney, head kidney, gills, spleen and intestine. The metabolites were extracted in methanol–chloroform and separated by normal-phase high-pressure liquid chromatography (HPLC) followed by scintillation counting. Identification of the metabolites was by comigration with standards on normal and reversed-phase HPLC systems and by protein-binding assays. All tissues from all species analysed produced hydroxylated derivatives identified as 25OHD₃, 24,25(OH)₂D₃ and 1,25(OH)₂D₃. In addition, some unidentified derivatives were recorded, one probably being 25,26(OH)₂D₃. Organs producing great amounts of one metabolite also produced considerable amounts of the other possible derivatives, suggesting a lower degree of specificity in fish organs than in human organs. The predominating metabolite was 24,25(OH)₂D₃ in all organs from salmon and mackerel during incubation with ¹⁴CD₃ and within most organs from all species during ³H25OHD₃ incubation. The latter observation probably results from the need for decreasing rather than increasing the calcium absorption in these species, which live at least some periods of life in a marine environment.     

Induction of Triploidy and Tetraploidy in Nile Tilapia, Oreochromis niloticus (L.)

Abstract.— Induction of triploidy and tetraploidy in Nile tilapia, Oreochromis niloticus , was investigated by heat shock, cold shock, hydrostatic pressure, and/or chemicals (cytochalasin A, B, and D). Additionally, efficacy of combined protocols was determined. Heat shock 10 min after fertilization induced triploidy when incubation temperature was 24 C but not when incubation temperature was 31 C. Heat shock of 40–41 C at 4–6 min after fertilization was effective in inducing up to 100% triploidy with hatchability similar to controls. Cold shock at 13 C for 45 min five min after fertilization induced 85–100% triploids. Heat shock and multiple heat shocking were the most effective treatments for the induction of tetraploidy. Two heat treatments of 41 C applied at 65 and 80 min after fertilization for 5 min each produced approximately 80% tetraploidy in hatched fry. Immersion of fertilized eggs in cytochalasin A, B, or D at concentrations up to 10 μg/L applied at various times and durations was ineffective in inducing triploidy or tetraploidy.