Days Post Hatching Research Articles

Food ingestion, consumption and selectivity of pompano,Trachinotus ovatus(Linnaeus 1758) under different rotifer densities

The growth, survival, food selection and consumption of pompano larvae under different rotifer densities as well as their colour preference during the rotifer feeding stage were examined in this study. Growth and survival of fish larvae were not significantly affected when rotifer density was between 10 and 20 mL−1. Fish larvae grew slower at 1 and 40 rotifers mL−1 than at 10 and 20 rotifers mL−1, and higher fish survival was achieved when fish larvae were exposed to 10 and 20 rotifers mL−1. The rotifer density of 1 mL−1 not only reduced food ingestion during the early stage, but also delayed diet switch from rotifer to copepod nauplii. On 5 days post hatching (DPH), larval pompano ingested more rotifers in dark-coloured tanks and ingested more rotifers when prey colour was green. Based on the results obtained in the present study, the culture of larval pompano larvae is recommended using dark wall tanks with a feeding density of 10–20 rotifers mL−1 during the initial feeding stage. This study proposes a management protocol to use appropriate type and quantity of live food to feed pompano larvae in a hatchery rearing condition, which could be applicable to the culture of fish larvae in other marine fish species.

Morphometrical Changes on Korean Rose Bitterling, Rhodeus uyekii, in Early Growth Period.

We investigated the process of yolk absorption in Korean rose bitterling, Rhodeus uyekii, and determined the changes in its morphometric characteristics. The R. uyekii from 1 days post hatching (DPH) to 21 DPH, the eye diameter (ED) was 5.4 at 5 DPH. Thereafter, the ED/total length (TL) ratio increased to 10.7 at 21 DPH (p<0.05). The yolk length (YL) decreased from 95.4 to 1.1 by 21 DPH, and this rate of decrease was greater than that for any other dimension (p<0.05). 12 morphometric dimensions/TL for the R. uyekii were measured at each sampling day from 21 DPH to 170 DPH. At just hatching, the average TL and BW were 6.1±0.09 mm and 4.9±0.07 mg, respectively. At 53 DPH, the average TL was 12.9±0.28 mm and the average BW was 14.7±0.72 mg; the total length growth equation was TL=5.507e0.038t (R2=0.916). Further, the body weight growth equation was BW=3.3647e0.0296t (R2=0.9354). The dimensions with regard to body depth showed the greatest growth rates in the external characteristics of the fish (p<0.05). The patterns of the morphometric characteristics measured in this study can be classified in three ways. The patterns were shown to be increased (y=0.0992x+ 12.07, R2=0.8333), decreased (y=–0.0569x+42.029, R2=0.8395) or maintained (y=0.005x+18.263, R2=0.3678) from 21 DPH to 170. These results will provide useful indices for the successful rearing of the R. uyekii.

Comparative Analysis of Histological Changes in Ussurian Bullhead, Leiocassis ussuriensis, and Korean Bullhead, Pseudobagrus fulvidevrepaco, in the Early Period of Growth.

The histological changes in the Ussurian bullhead, Leiocassis ussuriensis, and the Korean bullhead, Pseudobagrus fulvidevrepaco, were observed during the early period of growth. The retinas size of both species increased in the 9 days post-hatching (DPH) (p<0.05). In the just-hatched Ussurian bullhead, the retina already consisted of six layers: the epithelial layer, ganglion cell layer, inner nuclear layer, inner plexiform layer, outer limiting membrane layer, and rod and cone layer. The Korean bullhead had the same components. At 50 DPH, the thickness of the retina was 538.0±7.19 μm in the Ussurian bullhead and 558.9±9.44 μm in the Korean bullhead. The relative thickness of each layer of the retina did not differ significantly in the two species. Although the growth of the Korean bullhead’s retina was faster, the relative thickness of each layer in the retina did not change during early development. After hatching, some parts of the tissue gradually became denser. Immediately after hatching, the kidney and midgut epithelium of the Ussurian bullhead and Korean bullhead were already formed and grew gradually thereafter. From 0 DPH to 30 DPH, the nuclear height in the midgut epithelium did not differ significantly between the two species, but at 50 DPH, it was 11.4±2.45 μm in the Korean bullhead and 9.9± 2.13 μm in the Ussurian bullhead. During the experimental period, the major axes, minor axes, surface areas, and volumes of the proximal tubule cells in the kidney did not differ significantly between the two species. Thus, the early histological development of the Ussurian bullhead is similar to that of the Korean bullhead.

Morphometric and Histological Changes in Cyprinid Loach, Misgurnus anguillicaudatus, in the Early Growth Period.

In this study, we measured the morphometric and histological changes in the cyprinid loach, Misgurnus anguillicaudatus, during the early period of growth. Eyes, yolk length, yolk height, and yolk volume of the larva decreased for 16 days post hatching (DPH) (P<0.05). During 60 DPH (P>0.05), the most anterior extension of the head × the posterior end of the supraoccipital, the most anterior extension of the head × the origin of the dorsal fin, the most anterior extension of the head × the origin of the pectoral fin, the posterior end of the supraoccipital × the origin of the pelvic fin, and the origin of the dorsal fin × the ventral origin of the caudal fin gradually decreased, whereas the most anterior extension of the head × the dorsal origin of the caudal fin, the origin of the dorsal fin × the origin of the anal fin, the origin of the dorsal fin × the origin of the pectoral fin, and the insertion of the dorsal fin × the origin of the pelvic fin gradually increased (P<0.05). In the cyprinid loach, the retina is composed of six layers: the epithelial layer, ganglion cell layer, inner nuclear layer, inner plexiform layer, outer limiting membrane, and rod and cone layer (RCL). After hatching, part of the RCL gradually increased in density. The kidney and midgut epithelium were already formed in the cyprinid loach just after hatching and grew gradually in subsequent days.

Comparative Analysis of the Morphometric Changes in Ussurian bullhead, Leiocassis ussuriensis, and Korean bullhead, Pseudobagrus fulvidraco, in the Early Period of Growth.

Morphometric changes in the Ussurian bullhead, Leiocassis ussuriensis, and the Korean bullhead, Pseudobagrus fulvidraco, were observed during the early period of growth. Yolk length, yolk height, and yolk volume in the two species decreased within 9 days post-hatching (DPH) (p<0.05). The body lengths and body heights of both species increased gradually to 150 and 130 DPH, respectively (p<0.05). The horizontal distance between the anteriormost extension of the head and the anterior insertion of the pectoral fin, the anteriormost extension of the head × the verticality position of the anterior insertion of the primary dorsal fin rays, and the anterior insertion of the primary dorsal fin × the anterior insertion of the pectoral fin were greater in the Korean bullhead than in the Ussurian bullhead (p<0.05). However, the relative sizes of the head region, pectoral fin, ventral fin, and anal fin were greater in the Ussurian bullhead than in the Korean bullhead (p<0.05), and relative body depth and the size of the outer-mandible barbel were greater in the Korean bullhead than in the Ussurian bullhead (p<0.05). The growth curves of the morphometric characteristics of both species were divided into three types.

Effects of chronic aluminum and copper exposure on growth and development of wood frog (Rana sylvatica) larvae

Wood frogs (Rana sylvatica) were exposed to aluminum (Al; 10, 100, 500, 1000, or 2000μgL−1) or copper (Cu; 1, 10, 50, 100, 200μgL−1) at a pH of 4.70 from the beginning of the larval period through the completion of metamorphosis (range=43–102 days). Observations on mortality, malformation, time to reach specific developmental stages, body mass at these stages, and metamorphic success were made throughout the larval developmental period. Only one case of malformation was observed and mortality was ≤10% at all concentrations except the highest Cu concentration where the rate was 33%. All larvae that survived the experiment successfully completed metamorphosis, but significant effects on growth and development occurred for both metals and these were most prominent for Cu. At the highest Al concentration (2000μgL−1), body mass of larvae was significantly lower (reduced by 17% compared to the control) at 20 days post hatching (DPH) and the time to reach the hind-limb (HL), front-limb (FL), and tail resorption (TR) stages was significantly increased (9–10 days longer than the control). Body mass of larvae exposed to the three highest concentrations of Cu (50, 100, 200μgL−1) was reduced by 30–34% at 20 DPH. Exposure to these concentrations also resulted in increased time to reach the HL, FL, and TR stages with larvae in the highest concentration taking 21–29 days longer to reach these stages. Larvae exposed to 10μgL−1 Cu also took longer to reach the FL and TR stages of development, and exposure to all Cu concentrations increased tail resorption time by more than two days compared to the control. Although the only observed effects of Al were for a concentration that is probably not ecologically relevant, results demonstrate that environmentally-realistic levels of Cu may have significant biological effects that could influence individual fitness and population-level processes.

Body shape changes during the early development of the Beluga (Huso huso)

Early body shape changes of beluga sturgeon were studied using landmark-based geometric morphometric approach to recognize its allometric growth pattern. Sampling was done from hatching up to 50 days post hatching (DPH). Left side of specimens were photographed using digital camera and nine landmark points were digitized on two-dimensional images. Total length (TI) was measured using the software ImageJ. To study of the body shape changes during early development, the mean procrustes distance between all specimens of same age, for all age groups, was calculated. The scores of relative warp analysis (RW) were used as descriptors for the variation in shape. RW analysis revealed a sharp body shape change during early ontogeny on 18 DPH. Growth trajectory was computed by plotting RW against TL. The inflection point of body shape corresponds to a TL of 23.3 mm (18 DPH). Results showed that ontogenetic shape changes encompassed a pre-inflection shape changes, which included the elongation of the head and tail regions i.e. positive allometric growth pattern and post-inflection shape changes, with a nearly isometric growth pattern.

Changes in mineral concentrations in amberjack Seriola dumerili larvae during seed production: high concentrations of certain minerals in rotifers do not directly affect the mineral concentrations in larvae

This study examined the changes in mineral concentrations in amberjack Seriola dumerili larvae during seed production taking into consideration the mineral concentrations of rotifers in the larval rearing tanks (tank rotifers), since the concentrations of certain minerals in tank rotifers have been reported to be very high. In the present study, the concentrations of Ca, Mg, Fe, Zn and Mn in tank rotifers were generally higher than in freshly enriched rotifers. The extent of the increase in tank rotifers varied depending on the mineral, larval production facility and larval production trial. Concentrations of Ca in the larvae increased as they grew, those of P and Mn increased up to ca. 30 days post-hatching (DPH), those of Mg and Fe increased up to ca. 20 DPH and then decreased, and those of Zn continuously decreased through the seed production periods (to ca. 40 DPH). However, there were no clear relationships between the mineral concentrations of the tank rotifers and the larvae. These results indicate that high concentrations of minerals in tank rotifers are not directly reflected in amberjack larvae. This suggests that amberjack larvae maintain their mineral status by controlling the uptake and/or excretion of food minerals.

Metamorphosis Induces a Light-Dependent Switch in Senegalese Sole (Solea senegalensis) from Diurnal to Nocturnal Behavior

Light plays a key role in the development of biological rhythms in fish. Recent research in Senegal sole has revealed that spawning and hatching rhythms, larval development, and growth performance are strongly influenced by lighting conditions. However, the effect of light on the daily patterns of behavior remains unexplored. Therefore, the aim of this study was to investigate the impact of different photoperiod regimes and white, blue, and red light on the activity rhythms and foraging behavior of Solea senegalensis larvae up to 40 days posthatching (DPH). To this end, eggs were collected immediately after spawning during the night and exposed to continuous white light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white (LD(W)), blue (LD(B), λ(peak) = 463 nm), or red light (LD(R), λ(peak) = 685 nm). A filming scenario was designed to video record activity rhythms during day and night times using infrared lights. The results revealed that activity rhythms in LD(B) and LD(W) changed from diurnal to nocturnal on days 9 to 10 DPH, coinciding with the onset of metamorphosis. In LD(R), sole larvae remained nocturnal throughout the experimental period, while under LL and DD, larvae failed to show any rhythm. In addition, larvae exposed to LD(B) and LD(W) had the highest prey capture success rate (LD(B) = 82.6% ± 2.0%; LD(W) = 75.1% ± 1.3%) and attack rate (LD(B) = 54.3% ± 1.9%; LD(W) = 46.9% ± 3.0%) during the light phase (ML) until 9 DPH. During metamorphosis, the attack and capture success rates in these light conditions were higher during the dark phase (MD), when they showed the same nocturnal behavioral pattern as under LD(R) conditions. These results revealed that the development of sole larvae is tightly controlled by light characteristics, underlining the importance of the natural underwater photoenvironment (LD cycles of blue wavelengths) for the normal onset of the rhythmic behavior of fish larvae during early ontogenesis.

Development of Labrus Bergylta (Ascanius 1767) Larvae from Hatching to Metamorphosis

Ballan wrasse, Labrus bergylta (Ascanius, 1767), has commercial interest as a cleaner fish in aquaculture, and culture of juveniles may be a good alternative to catching wild fish. However, basic knowledge of early development is limited. To study hatching and larvae ontogenesis, eggs were collected from a broodstock of wild caught ballan wrasse held in captivity. Larvae were fed rotifers for 27 days; Artemia was introduced at day 20 and fed larvae to day 49 after hatching. Age at hatching, and larval ontogenesis based on external morphological features were linked to the chronological age of larvae as days post hatching (DPH) and physiological age as degree day (°C.day) and standard length (SL). The ballan wrasse egg diameter was 1.05 ± 0.04 mm and 0.87 ± 0.05 mm with and without the gelatinous layer, respectively, and hatched at a SL of 3.64 ± 0.05 mm, 7 days, i.e., 72°C.day, after fertilization. The ontogeny of larvae from hatching to metamorphosis was divided into four stages. Yolk sac larva, 0 to 9 DPH (SL 4.28 ± 0.11 mm), where mouth opens and pigmentation of the eyes is seen. Dilated swim bladder is visible. Preflexion larva, 10 to 25 DPH (SL 5.35 ± 0.30 mm); yolk sac has disappeared, and initial formation of caudal fin rays occurs. Gaseous inflation of swim bladder is observed. Flexion larva, 26 to 33 DPH (SL 5.9 ± 0.78 mm), initial resorption of primordial fin fold is seen. Postflexion larva, 34 to 49 DPH (SL 10.52 ± 0.82 mm) by the end of this stage (age 686°C.day), the dorsal, anal, caudal and pelvic fins were developed. The ontogenetic development, linked to SL, chronological and physiological age, provides a baseline reference and makes it possible to compare developmental levels in future studies of ballan wrasse in culture and wild populations.

Breeding of the forktail blenny Meiacanthus atrodorsalis: Broodstock management and larval rearing

The present study describes the reproductive behavior of the forktail blenny, Meiacanthus atrodorsalis, and investigates the survival and growth of larvae under various feeding regimes. Two harems of six M. atrodorsalis began spawning about 3 months after acquisition, with egg clutches discovered regularly throughout the study period. Observations of courting behavior suggested that the female initiated courtship with spawning commencing after a series of male displays and courtship encounters. Adult M. atrodorsalis displayed a preference for shelters of single entrance 50-mm PVC pipe, with a 25-mm reduced entrance, for egg laying, while the male took full responsibility for egg care. Larvae measuring 3.11 ± 0.15 mm standard length (SL) and 0.63 ± 0.03 mm body depth (BD) hatched approximately 181 h post fertilization at 28 °C, with a mouth gape height and width of 307.3 ± 11.0 and 263.8 ± 5.5 μm, respectively. Using a feeding protocol consisting of firstly feeding rotifers, and then switching to Artemia nauplii with enriched Artemia meta-nauplii added later on, larvae were observed to reach settlement approximately 35 days post hatching (DPH), measuring 13.54 ± 0.38 mm SL and 3.91 ± 0.27 mm BD. The first feeding trial tested the effect of rotifer density on survival and growth of newly hatched larvae. The results showed no significant difference in either larval survival or growth when fed rotifers at 2, 5, 10 and 20 rotifers mL −1. The second trial investigated the appropriate time to shift from feeding larvae with rotifers (10 mL −1) to Artemia nauplii (3 mL −1). These feeding trials suggest that to optimize survival and growth, newly hatched larvae of M. atrodorsalis should be fed rotifers at a density of between 2 and 20 rotifers mL −1, and that the majority of larvae appeared to display the ability to ingest Artemia nauplii between 6 to 10 DPH.

Exposure of larvae to daily thermocycles affects gonad development, sex ratio, and sexual steroids in Solea senegalensis, kaup

The effect of water temperature during the development of fish larvae on sex differentiation is well known, but not so well known is the impact of the daily thermocycles. Our aim was to investigate the effect of early exposure of Senegal sole larvae to different temperature cycles on gonad development, sex ratio, and sex steroid (11-ketotestosterone (11-KT); estradiol (E(2) ); and testosterone, (T)) content in muscle extracts of juveniles. From 1 to 97 days posthatching (DPH) fish larvae and post-larvae were subjected to three temperature regimes: Thermophase-Cryophase (TC), Cryophase-Thermophase (CT), and constant temperature. In fish exposed to TC, sex determination occurred earlier, because 90% of soles were males/females at 110 DPH, whereas 45% of fish under CT were undifferentiated at that time. Fish under TC showed the highest growth rates, followed by fish under constant temperature and by fish under CT, the differences being statistically significant between the TC and CT groups. Regarding sex ratio, juveniles exposed to TC showed a higher proportion of females than fish under CT or constant temperature. Under TC, fish showed the highest concentration of E(2) , whereas 11-KT concentration was highest in fish under CT and constant temperature. Fish under constant temperature and CT showed higher T levels than those under TC. These results provide the first insights into the effect of daily thermocycles on sex differentiation in fish, and underline the key role of natural environmental cycles on the control of sex ratios during larval development, which may be applied to the manipulation of sex ratio in aquaculture.

Effects of dietary n-3 HUFA concentrations on spawning performance and fatty acids composition of broodstock, eggs and larvae in yellowfin sea bream, Acanthopagrus latus

We conducted a study to investigate the effects of dietary n-3 highly unsaturated fatty acid (HUFA) concentrations on spawning performance and fatty acids composition of broodstock, eggs and larvae of Acanthopagrus latus. Broodstock were fed three isonitrogenous formulated diets with different ratios of fish oil (FO) and sunflower oil (SO) including FO (100% fish oil), FSO (1:1 fish oil: sunflower oil) and SO (100% sunflower oil) as lipid sources with different concentrations of n-3 HUFAs (6.67, 4.26 or 2.92%, respectively) for 132 days. Results show that the relative fecundity, percentage of buoyant eggs, hatchability, survival rate of larvae at 3 days post hatching (DPH) and starvation tolerance test were all higher in broodfish fed FO diet and significantly (P < 0.05) decreased in the groups fed FSO and SO diets. Fatty acids profiles of carcass, liver, eggs, hatchling and 3DPH larvae reflected profiles of the corresponding dietary treatments. In general, increasing concentrations of dietary fish oil lowered deposition of linoleic acid (LA), linolenic acid (LNA) and total n-6 PUFA in all samples and simultaneously improved deposition of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (AA) in all samples, which probably affected spawning performance. The best spawning performance was obtained when fish oil was used alone as the dietary lipid source with 6.6% dietary n-3 HUFA concentration. The results of this study show that the n-3 HUFA concentrations of lipid in broodstock diet has a considerable effect on the quality and fatty acids composition of egg and larval in A. latus.

Sex-related changes in estrogen receptors and aromatase gene expression and enzymatic activity during early development and sex differentiation in the European sea bass ( Dicentrarchus labrax)

The present study addresses the role of aromatase and estrogen receptors in sex differentiation and development. With this purpose, a sea bass female- and a male-dominant group were obtained by successive size gradings since in this species females are already larger than males at the time of sex differentiation. Changes in cyp19a and cyp19b gene expression and enzymatic activity were monitored by a validated real-time PCR and a tritiated water assay, respectively, during early development and sex differentiation. Changes in mRNA expression of estrogen receptors, both erb1 and erb2, were also assessed during this period. Results show clear sex-related differences in cyp19a gene expression and enzymatic activity in gonads, with females exhibiting significantly higher levels than males at 150 days post hatching (DPH), when histological signs of sex differentiation were evident. cyp19b gene expression and activity in brain were detectable during early ontogenesis at 50 DPH but no clear sex-related differences were observed. Both erb1 and erb2 showed higher gene expression levels in testis than in ovaries around 200–250 DPH, corresponding with the time of testicular differentiation and precocious male maturation, but no sex-related differences were found in the brain. Together these results indicate that in the European sea bass high expression levels of cyp19a are associated with ovarian differentiation and thus cyp19a can be considered as a suitable molecular marker of ovarian differentiation. However, the involvement of cyp19b in sex differentiation cannot be concluded. In addition, the higher levels of erb1 and erb2 in males versus females during sex differentiation, coinciding with precocious male maturation in the sea bass, suggest an important role for these receptors in testicular development and maturation.

Changes in RNA/DNA ratio and growth of slime flounder, Microstomus achne, larvae until metamorphosis

Summary Understanding the nutritional condition and survival of fish larvae is of primary importance in mass larva culture because intensive mortality is concentrated during the larval period. In order to estimate growth and nutritional status based on biochemical indices of slime flounder, Microstomus achne, larvae reared under starved and fed conditions in the hatchery for 58 days and the changes of RNA, DNA, and protein contents were described with the progress of growth and developmental. DNA contents increased gradually throughout the experimental period until 12 and 58 days post-hatching (DPH) in starved and fed groups respectively. Although they fluctuated and decreased around 12 and 46 DPH, the RNA contents of the fed group increased gradually after hatching; however, in the starved group, they decreased after yolk absorption and 7 DPH. Subsequently, the RNA/DNA ratios in the starved group remained constant until 6 DPH and then decreased rapidly. In the fed group, this decreased slightly from hatching to 14 DPH, then increased gradually until the end of the experiment, except at the lower level of around 46 DPH. Namely, temperature shocks (around 14 DPH) and the dramatic changes in body shape (around 46 DPH) were accompanied by the decrease of the RNA/DNA ratios. Total protein/DNA in both groups decreased rapidly during yolk absorption 0–7 DPH, then decreased continuously until death in the starved group; in the fed group total protein/DNA increased again with feeding. It is suggested that the changes in these biochemical values reflect yolk absorption, feeding, morphogenetic changes, growth, and environmental conditions.

Deformities in larval gilthead sea bream ( Sparus aurata): A qualitative and quantitative analysis using geometric morphometrics

Deformities in commercially raised fish are a common source of downgrading of product value. During the intensive rearing of gilthead sea bream ( Sparus aurata), opercular deformities are the most commonly observed type of deformation (affecting up to 80% of the fisheries stock), sometimes showing a severe inward folding of the operculum. They are non-lethal malformations that appear during the larval stage but affect growth rate and morphology, with a significant economic loss as a consequence. In order to exploratory quantify and qualify these deformities, geometric morphometric analyses were performed on the external morphology from larvae with an age ranging from 50 to 69 days post-hatching (DPH), and on the cranial skeleton of 110 DPH old juveniles. The results showed several osteological cranial shifts and a striking left–right independency associated with deoperculation. Even though a significant size difference was observed at 65 DPH between normal and deoperculated specimens, allometries during the examined growth stages still appear to be very similar in normal and deoperculated specimens. At 69 DPH deoperculated specimens differed significantly from the normal specimens in their external morphology based on its shape variables, but the results suggest that discrimination is possible from earlier stages. Further analyses are needed, but the usefulness of this approach towards developing an early detection tool could be demonstrated.

The larval rearing of the marine ornamental crab, Mithraculus forceps (A. Milne Edwards, 1875) (Decapoda: Brachyura: Majidae)

The goal of this study is to develop a larviculture protocol for Mithraculus forceps, a popular marine aquarium species. Different temperatures (25±0.5°C and 28±0.5°C), stocking densities (10, 20, 40 and 80 larvae L−1), prey densities (newly hatched Artemia of 1, 4, 7 and 12 nauplii mL−1) and metamorphosis to crab conditions (Systems A and B) were tested. The best survivorship and faster development were obtained when the larvae were reared at a density of 40 larvae L−1 for 7 days post hatching (DPH) in System A, at 28°C and fed with 7 mL−1 of newly hatched Artemia nauplii. After 7 DPH all the megalopa were moved to System B and the same temperature and prey density were maintained. At the end of the experiment, 12 DPH, survivorship of 74.1±4.8% was obtained.

Sex reversal in Nile tilapia (Oreochromis niloticus Linnaeus) by androgen immersion

Two experiments were conducted to investigate the efficacy of three androgens applied through immersion treatments on the sex ratio of Nile tilapia (Oreochromis niloticus L.) fry. In experiment 1, 14 days post-hatching (DPH) larvae were exposed to a single immersion treatment in 17α-methyltestosterone (MT), 17α-methyldihydrotestosterone (MDHT) or 17α-ethynyltestosterone (ET) at 200, 600 and 1800 μg L−1 over 4 h (130 larvae per treatment). In experiment 2, Nile tilapia larvae were exposed to the higher androgen concentration (1800 μg L−1) applied as either a single immersion (14 DPH) or double immersion (10 and 14 DPH) over 4 h (125 larvae per treatment). Change in sex proportion within each experiment as well as between experiments was analysed by the chi-square test. In experiment 1, MT, MDHT and ET were equally effective in significantly increasing the proportion of males when applied at 1800 μg L−1 (86.0%, 90.0% and 86.7% respectively). At 200 μg L−1 none of the androgens altered sex ratio. At 600 μg L−1, only MDHT slightly, but significantly skewed the sex ratio towards males (73.0%). In experiment 2, a single immersion treatment at 14 DPH (1800 μg L−1) significantly increased the proportion of males, but at this time the response was significantly hormone dependent (MDHT, 100.0%; MT, 91.6%; ET, 76.9%). When compared with a single immersion, two-immersion treatments significantly increased the proportion of males in the MT-treated group (from 91.6% to 98.3%), decreased the proportion of males in the MDHT group (from 100.0% to 93.4%) and had no significant effect the ET-treated group (change from 76.9% to 82.5%). The overall comparison of the sex ratio among same treatments from different experiments (a single immersion in 1800 μg L−1) was not significantly different.

Y-chromosomal DNA markers for discrimination of chemical substance and effluent effects on sexual differentiation in salmon.

Chinook salmon alevins were exposed during their labile period for sex differentiation to different concentrations of bleached kraft mill effluent (BKME), primary sewage effluent, secondary sewage effluent (SE), 17ss-estradiol, testosterone, and nonylphenol. After exposure for 29 days post hatching (DPH), fish were allowed to grow until 103 and 179 DPH, at which time their genetic sex was determined using Y-chromosomal DNA markers and their gonadal sex was determined by histology. Independent of treatment, all fish identified as genetic females (XX) in these experiments possessed normal female gonads. Only the highest dose of some treatments affected the development of gonads in genetic XY males. At 103 DPH, some genetic males treated with 1 micro g estradiol/L, BKME 100%,and SE 30% developed as physiological females, presenting ovaries identical to genetic females in the control group. The physiological female condition in XY fish was also observed in these treatments groups at 179 DPH, which suggests that the effect is permanent, whereas in other groups the effect changed between sampling periods. Identification of the genetic sex of individual animals using sex-linked DNA markers provides a useful tool for investigating environmental factors influencing sex determination and differentiation.

Effects of n−3 HUFA levels in broodstock diet on the reproductive performance and egg and larval quality of the Japanese flounder, Paralichthys olivaceus

We investigated the effect of dietary n−3 highly unsaturated fatty acids ( n−3 HUFA) on egg and larval quality in the Japanese flounder Paralichthys olivaceus. Diets with similar proximate composition, but different n−3 HUFA (0.4%, 0.8%, and 2.1%) levels were fed to broodstock from 3 months before and during the spawning season. Spawning was observed from March to June. Total egg production was highest in fish fed the lowest n−3 HUFA, and decreased with the increase of dietary n−3 HUFA. Percentages of buoyant eggs and hatched larvae were independent of dietary treatment. In contrast, the percentage of normal larvae, survival of larvae at 3 days post hatching (DPH), and the starvation tolerance index correlated with dietary n−3 HUFA levels. n−3 HUFA level in eggs increased with the increase of dietary level. Higher level of arachidonic acid (AA) was also found in eggs that showed high quality, reflecting dietary fatty acids. The results of this study clearly show that n−3 HUFA level in broodstock diet has an effect on the quality of eggs and larvae in the Japanese flounder and suggested that AA may have positive effect on the improvement in spawning quality of flounder.