The purification and development of a quantitative enzyme linked immunosorbent assay (ELISA) for the measurement of vitellogenin in diploid and triploid brook trout (Salvelinus fontinalis)

Vitellogenin (VTG) was purified from the plasma of estradiol-17β (E₂) treated male brook trout (Salvelinus fontinalis; bt) using gel filtration and anion exchange chromatography. An antiserum to bt-VTG was raised in rabbits, then used to detect bt-VTG by Western blot analysis. Purified bt-VTG and its antibody were then used to develop an antibody-capture, competitive enzyme-linked immunosorbant assay (ELISA). Confirmation of the purified protein as bt-VTG was based on the characteristics of high molecular weight (∼562 kDa), dominant plasma protein in vitellogenic females and induction by exogenous E₂ treatment in males. The antiserum recognized a major 184 kDa polypeptide as well as minor bt-VTG degradation products (150–66 kDa) in purified bt-VTG and in plasma from vitellogenic females. Low levels of antibody cross reactivity were shown with plasma from nonvitellogenic females, control rabbit serum, and several other antisera known to not contain VTG. The ELISA had a minimum detection limit of 8 ng ml⁻¹ and intra- and inter-coefficients of variation less than 9% and 15%, respectively. The ELISA demonstrated parallel binding slopes among dilution curves of purified bt-VTG standard and plasma from diploid and triploid brook trout females. Using the ELISA, maximum plasma VTG levels of 93.5±33.6 mg ml⁻¹ were detected in vitellogenic diploid females, whereas only 0.18±0.15 mg ml⁻¹ were detected in triploid females of the same age (n=5 for each). Diploid and triploid males cohabitating with vitellogenic females showed measurable levels of plasma VTG during vitellogenesis in females (i.e., 0.17 and 0.06 mg ml⁻¹, respectively (n=1)). This revised version was published online in August 2006 with corrections to the Cover Date.     

Gag (Mycteroperca microlepis) vitellogenin: purification,characterization and use for enzyme-linked immunosorbent assay (ELISA) of female maturity in three species of grouper

Circulating levels of the egg yolk precursor protein, vitellogenin (VTG), can be used as a biochemical indicator of maturation in female fish. Here we report on purification and partial characterization of VTG from a temperate marine serranid, the gag(Mycteroperca microlepis). Development of a competitive, enzyme-linked immunosorbent assay (ELISA) for gag VTG (gVTG) is also described. The gVTG was purified by DEAE-agarose anion exchange chromatography from a pooled plasma sample collected from several juvenile gag after they were injected with 17estradiol. The protein appeared as a major band of M_r183000 after SDS-PAGE ± Western blotting using either a specific rabbit antiserum to gVTG or a universal monoclonal antibody for vertebrate VTGs. Amino acid composition analysis and N-terminal peptide sequencing verified that gVTG is similar in primary structure to VTG from several other teleost species. The purified gVTG and its specific antiserum were used to develop a sensitive, competitive, antibody-capture ELISA for quantifying the protein in blood plasma from maturing females. VTG levels in maturing female gag were highly correlated with oocyte growth and circulating testosterone and 17-estradiol levels, whereas VTG was non-detectable in juveniles, immature females or males. Two size-based maturity schedules for female gag were constructed, one utilizing detection of VTG in their circulation as a marker of maturity and the other relying on histological evidence that their ovaries were in vitellogenic or later stages of maturation. The two schedules were virtually identical. The gVTG ELISA was also used to detect VTG in blood plasma from mature Nassau grouper (Epinephelus striatus) and red hind (E. guttatus). As with gag, the assay was completely reliable for discriminating between reproductively mature females versus males from these two grouper species.     

Disparate effects of gonadal steroid hormones on plasma and liver mRNA levels of insulin-like growth factor-I and vitellogenin in the tilapia, Oreochromis mossambicus

The tilapia, Oreochromis mossambicus, exhibits a sexually dimorphic pattern of growth, males growing larger than females. We examined the effects of E₂ and DHT on the GH/IGF-I axis and on VTG production in the tilapia. Sexually mature tilapia were injected with 5 μg g body weight of E₂ (males) or DHT (females) every 5 days for a total of 3 injections. Female tilapia had significantly higher plasma GH levels than males. However, plasma and liver mRNA levels of IGF-I were significantly lower in females than in males, whereas VTG levels in both the plasma and liver mRNA were significantly higher in females than in males. Although significant amounts of VTG were detected in control males (8 ± 0.3 μg ml), the levels in control females (3000 ± 500 μg ml) were about 400 times higher than in males. Males treated with E₂ exhibited a female-like GH/IGF-I profile. That is, they had significantly elevated levels of plasma GH with lower plasma IGF-I and liver IGF-I mRNA levels. Estradiol treatment significantly elevated both plasma and liver mRNA VTG levels. Dihydrotestosterone treatment in females induced a male-like GH/IGF-I profile: plasma GH levels were significantly reduced, whereas plasma and liver IGF-I mRNA levels were significantly elevated. Both plasma and liver mRNA levels of VTG were not altered by DHT treatment. Pituitary GH mRNA levels were similar in all treatment groups. These results clearly indicate that estrogens and androgens feminize and masculinize the GH/IGF-I axis, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Photoperiodic manipulations of the reproductive cycle of sea bass (Dicentrarchus labrax) and their effects on gonadal development, and plasma 17ß-estradiol and vitellogenin levels

The annual profile of plasma vitellogenin (VTG) and 17ß-estradiol (E₂) levels, as well as gonadal development and spawning characteristics were investigated in captive female sea bass (Dicentrarchus labrax). Endocrine and gonadal changes were studied in fish reared under natural conditions or exposed to manipulated photothermal cycles. In natural conditions of photoperiod and temperature, sea bass spawned from February through March (East coast of Spain, 40°N 0°E). One or two months of constant long-days (15L/9D) in a constant short-day photoperiod regime (9L/15D) all-year-round, given early in the year (March and March–April), advanced spawning by 3 months. The same treatment applied later in the year (September–October) delayed spawning by 1 month, compared to controls.In all groups, changes in plasma VTG levels were correlated with E₂ levels, oocyte growth and spawning time. In control females, VTG was low (<100 ng ml^-1) during the summer, until its first surge in plasma 4 months before the beginning of spawning. The VTG (3.1 ± 0.3 mg ml^-1) and E₂ (4.1 ± 0.5 ng ml^-1) levels showed a single annual peak during late vitellogenesis, the time of the highest proportion of vitellogenic oocytes in the ovary. Constant high levels of VTG (1–1.4 mg ml^-1) and E₂ (1.6–1.9ng ml^-1) were maintained during the entire spawning time, together with the presence of vitellogenic oocytes, suggesting the existence of several waves of oocyte growth in the ovary and thus, several spawns per female. Endocrine profiles and oocyte development in fish exposed to constant photoperiods were similar to controls, but were shifted in time in relation to the displacement of the spawning time. In the fish showing advanced spawns, the duration of the gametogenic proces was compressed when compared to controls. The differences observed in the evolution of the reproductive-related factors in the advanced groups, which were exposed to a reduction in temperature to 15°C, suggest an influence of the temperature in the early stages of the reproductive cycle in sea bass.     

The modulating effect of vitellogenin on the synthesis of 17β-estradiol by rainbow trout (Oncorhynchus mykiss) ovary

In vivo induction of vitellogenin (VTG) in response to the administration of 17-estradiol (E₂) was achieved and the protein was isolated by gel filtration column chromatography of plasma samples. Adult female trout were injected with the vitellogenic fraction every ten days from July to November and levels were measured by RIA from September to December. The results showed a significant decrease (p<0.01) in plasma E₂ levels in injected females compared with the controls. In December, after finishing the treatment, the plasma E₂ concentration increased, in injected females to reach a level similar to that of control females at vitellogenesis. The in vitro study showed that in early vitellogenic oocyte (from September) the presence of the vitellogenic fraction in the incubation medium causes a significant decrease (p<0.01) in the synthesis of E₂ by the oocytes. These data suggest that the concentration of the VTG into the oocyte can alter VTG production by the liver, moderating the production of E₂ by the ovary.     

Relationship between metabolic and reproductive hormones in salmonid fish

Circulating concentrations of estradiol (E₂), vitellogenin (VTG), thyroxine (T₄), triiodothyronine (T₃) and insulin were measured in reproductively maturing four and five year-old Atlantic salmon. Blood samples were collected from the fish in seawater for one year prior to their spawning in November in fresh water. In females, E₂ and VTG were low but detectable from December to July, and then increased to peak levels in September and October. Plasma levels of T₄ and T₃ were relatively constant in winter and spring, and decreased in July. Plasma concentration of T₄ increased in November when the fish returned to fresh water. Plasma T₃ levels remained low during the autumn. Both T₄ and T₃ levels tended to be higher in males than in females during September through November. Plasma insulin concentrations increased during the spring to peak values in May, and then decreased in June and July in fish of both sexes. There was a significant elevation of plasma insulin in males during October, and the levels in males tended to be higher than those found in females during final maturation.     

Atlantic halibut (Hippoglossus hippoglossus) vitellogenin: induction,isolation and partial characterization

Vitellogenin (VTG) synthesis was induced by repeated injections of estradiol-17 in juvenile Atlantic halibut (Hippoglossus hippoglossus). VTG eluted as a large, phosphoprotein containing peak on DEAE-Sephacel chromatography of plasma from estradiol-17 treated juvenile and mature female, but not mature male halibut. A purification procedure for Atlantic halibut VTG was developed, where VTG was precipitated with MgCl₂, EDTA and distilled water, and the precipitated protein submitted to anion exchange chromatography on DEAE-Sephacel. Precipitated VTG eluted as a broad, partly dissociated peak on DEAE-Sephacel, when chromatography was run at 4°C, but the protein appeared intact when analysed both by SDS PAGE and native PAGE. DEAE-Sephacel chromatography at room temperature resulted in an irregular elution pattern and a dissociated protein fraction, as analysed by SDS PAGE. Biochemical characterization of VTG showed that the molecular mass of the monomer was ca 160 kDa, as estimated by SDS-PAGE. The total lipid content was 19.8% w/w, with 64%, or 12.7% of the total weight, as phospholipid. Protein bound phosphorus constituted 0.62% w/w of halibut VTG. Plasma dilution curves from mature and maturing female halibut were parallel with a dilution curve from halibut egg yolk homogenate in an homologous RIA. Plasma from mature male, but not juvenile halibut crossreacted with the VTG antiserum.     

The prevalence of mycobacterial infections in striped bass in Chesapeake Bay

Several species of bacteria have been isolated from diseased striped bass, Morone saxatilis (Walbaum), in Chesapeake Bay. Mycobacteria species are of particular interest because of their association with chronic lesions in both fish and humans. The resulting disease in fish may be characterised by emaciation, inflammation of the skin, exophthalmia (pop‐eye), ascites (dropsy), open lesions, and ulceration. The prevalence of mycobacterial infection in 217 striped bass in the Maryland portion of Chesapeake Bay was determined to provide evidence of decreased overall condition of fish affected by these bacteria. The density of the granulomas was higher in the spleen and head kidney than either the heart or liver, and appeared to be most closely associated with the presence of an external lesion. Thirty‐eight per cent of the fish showed some sign of an external lesion. Granulomas appeared in at least one organ in 53% of the fish sampled regardless of the presence or absence of an external skin lesion. Condition factor was significantly higher for striped bass without external lesions (0.96) than those with lesions (0.82). Striped bass with no internal granulomas and no external lesion had the highest condition factor (0.99) and fish with granulomas and external lesions the lowest (0.81). Slopes of the regression log_e weight (g) and log_e length (mm) for striped bass with external lesions was significantly higher than those fish without external lesions and had a lower r². The results suggest that striped bass that are infected with mycobacteria have decreased overall health.     

The Annual Reproductive Cycle of the White Bass Morone chrysops

The gametogenic cycle of the white bass Moronc chrysops was characterized for one year at monthly intervals by measuring circulating levels of the sex steroid hormones testosterone (T) and 11-ketotestosterone (11-KT) in males, and 17β-estradiol (E₂) and T in females. Vitellogenin (VTG), the egg yolk precursor, was also measured in female blood plasma. Gonadal status of individual fish was assessed by histological evaluation, measurement of oocyte diameters, and spermintion response. At the onset of vitellogenesis (October), blood plasma levels of E₂ and T were low, but increased significantly as the spawning period (March-April) approached and oocytes attnined their maximum diameter. All stages of oocyte development were observed in mature females, indicating that the white bass is a multiple-clutch, groupsynchronous spawner. Androgen levels increased in males throughout the reproductive cycle and reached maximal levels during the spawning period. The increase in androgens coincided with sperm production and spermintion in males. The circannual endocrine and gonadal development patterns observed in white bass were similar to those of other members of genus Moronc .     

Effects of Natural EstradioI-17β and Synthetic 17α- Ethynylestradiol on Direct Feminization of European Sea Bass Dicentrarchus labrax

To produce a monosex female population of European sea bass Dicentrarchus labrax, fry were fed dry diets containing dosages of 12.5, 25, and 50 mg/kg food of either the natural estrogen estradiol-170β(E₂) or the synthetic estrogen 17α-ethynylestradiol (EE₂) for 60 d starting at 88 d post-hatch (dph). A complete feminization (100%) was achieved in all E₂-treated groups at the age of 11 mo (330 dph). All affected fish had ovaries similar in size and histological structure to those of control females. In the E₂-treated groups, feminized fish were heavier and longer than untreated controls (males and females combined). In control groups females exhibited significantly higher body weight and total length than males. Untreated females from control groups and females from the group treated with E₂ at 12.5 mg/kg food had similar body weight, suggesting that in sea bass growth is related to phenotypic sex. In the Entreated groups, survival rates were similar to those of the control fish. A relatively high percentage of females was obtained in the EE₂-treated groups (from 38.6 to 96.5%). However, the gonadal development in these fish was significantly suppressed and a dose-dependent reduction of gonadal sizes was evident. Treatments with the EE₂ (12-5, 25, and 50 mg/kg food) resulted in many fish having abnormal (2.9-5.4-39.8%, respectively) and sterile (0.6-6.0-21.6%, respectively) gonads. Effects also included significantly lower weight and shorter length when compared with controls. Furthermore, fish fed with EE₂ at the dosage of 50 mg/kg food had high mortality rate. A simple protocol was developed for the complete feminization in sea bass in which the fry (80-100 dph) were fed to satiation two times daily with a diet containing 12.5 me of E₂/ks food for a period of 60 d.     

Acute and Sublethal Effects of Ammonia on Striped Bass and Hybrid Striped Bass

Bioassays in static water (mean ± SD; temperature, 20–22 C; pH, 8.2–8.4; alkalinity, 205 ± 10 mg/L CaCO₃; total hardness, 220 ± 10 mg/L CaCO₃) were used to determine median lethal concentrations (LC₅₀) of un-ionized ammonia (NH₃-N) for striped bass Moronc saxatilis and hybrid striped bass M. saxatilis × M. chrysops. The 96 h LC₅₀ for striped bass was 1.01 ± 0.24 mg/L NH₃-N₃ and was significantly higher than the LC₅₀ for hybrid striped bass (0.64 ± 0.05 mg/L NH₃-N). The effects of sublethal ammonia were evaluated after fish were exposed for 96 h to 0.0, 0.25, or 0.5 mg/L NH₃-N or to additional exposure to oxygen depleted water (about 2.0 mg O₂/L). Plasma ammonia of striped hass did not change as sublethal ammonia increased, but exposure to oxygen depletion caused a decrease in plasma ammonia. In contrast, plasma ammonia of hybrid striped bass increased as environmental ammonia increased, and increased further after exposure to oxygen depletion. Plasma cortisol levels of striped bass were significantly higher and more variable than cortisol levels of hybrid striped bass; additional exposure to oxygen depletion increased this variability, but these responses may be due to the stress of handling and confinement. Mean differences also existed between species for hemoglobin and hematocrit, while differences in variability occurred for osmolality and oxygen depletion rates. Striped bass tolerated ammonia better than hybrid striped bass but were more susceptible than hybrid striped bass to the additional stress of oxygen depletion. Most changes in physiological characteristics were relatively independent of environmental ammonia, but they were affected by oxygen depletion challenge.     

Purification and partial characterization of vitellogenin from spotted wolffish (Anarhichas minor) and development of an enzyme-linked immunosorbent assay for the determination of gender and sexual maturity

Vitellogenin (VTG) from spotted wolffish, Anarhichas minor, a candidate species for cold-water marine aquaculture, was purified by MgCl₂/EDTA precipitation followed by a two-step chromatographic procedure. VTG had an apparent molecular mass of 470 kDa, as determined by gel filtration, and an amino acid composition similar to those of other teleosts. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the purified VTG revealed a major band with a relative molecular weight of 166 kDa and some minor bands. Spotted wolffish VTG (sw-VTG) is relatively robust to in vitro degradation, as shown when samples of purified VTG and plasma from mature females subjected to various storage conditions or multiple freeze/thaw cycles were analyzed by Western blot. We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) using an antibody against Atlantic wolffish (Anarhichas lupus) VTG and purified sw-VTG. The ELISA had a detection limit of 6.7 ng/ml and a working range of 16.2–787.5 ng/ml, with intra- and inter-assay coefficients of variation ranging from 1.5 to 7.3 % and 7.1 to 14.3 %, respectively. The assay could distinguish males from immature females and discriminate maturing females at different stage of oocyte development. These results suggest that the sw-VTG ELISA would be useful in spotted wolffish aquaculture to determine sex and monitor female maturation.     

Salinity and temperature effects on whole-animal thyroid hormone levels in larval and juvenile striped bass,Morone saxatilis

Whole-animal thyroxine (T₄) and 3,5,3′-triiodothyronine (T₃) levels were measured in larval and juvenile striped bass, Morone saxatilis, reared for 10 days at one of three levels of salinity (equivalent to fresh water (FW), one-third seawater (1/3 SW), and seawater (SW) and two temperatures (15°C and 20°C). The striped bass were pre-metamorphic larvae, metamorphic larvae or juveniles. The short-term effects of seawater on plasma T₄ levels of juvenile striped bass were also measured. Higher salinities increased T₄ levels in premetamorphic larvae. In metamorphic larvae, SW and 1/3 SW increased T₄ levels and SW increased T₃ levels at 20°C. This response was eliminated in those at 15°C. Whole-animal thyroid hormone content was unaffected by salinity or temperature in juvenile striped bass, although significant fluctuations in plasma T₄ levels occurred in those transferred to 1/3 SW and SW. The thyroid axis of striped bass responds to salinity and temperature as early as in the pre-metamorphic stage. Thyroid hormones may mediate the beneficial effects of salinity on larval striped bass growth and survival.     

Annual cycle of plasma lipids in captive reared striped bass: effects of environmental conditions and reproductive cycle

Protein, lipid and fatty acid concentrations in plasma of eight male and eight female six year old captive striped bass (Morone saxatilis) were monitored monthly over the course of two reproductive cycles as part of an effort to investigate the time course of lipid class mobilization and subsequent deposition in gonads. Total protein levels (44.2 ± 0.67 mg ml^–1, range 20.6–86.4) showed seasonal fluctuation, but did not vary with sex. Total lipid concentrations in the plasma of both males (17.7 ± 0.61 mg ml^–1) and females (14.1 ± 0.64 mg ml^–1) showed seasonal fluctuations with the lowest levels in late Spring during spawning. Plasma lipids in females were significantly (p < 0.0001) lower than those of the males except during early ovarian secondary growth. Analysis of the lipid class composition revealed that the decrease in plasma lipid concentrations in females prior to spawning is primarily due to a decrease of up to 50% in the phospholipid content of the plasma relative to males. Striped bass vitellogenin was found to contain approximately 20% lipid by weight with nearly 80% of the lipid being phosphatidyl choline (PC). Vitellogenin levels previously measured in these individuals were highest during the winter months and decreased in the 2 months prior to spawning. Although mature striped bass oocytes are rich in wax esters, no wax esters or fatty alcohols were found in the plasma. Analysis of the fatty acyl composition of separated lipid classes suggests that PC is the primary carrier of the essential (n=3) fatty acids 22:6 (DHA) and 20:5 (EPA) to the gonads. Fatty acid analysis of the lipids associated with vitellogenin revealed that levels of essential fatty acids were low relative to vitellogenin-borne lipids from other teleosts. The lack of detectable wax esters in plasma lipids of female striped bass suggests that these abundant lipids in mature eggs are synthesized within the oocytes.     

Vitellogenin in tilapia (Oreochromis mossambicus): Induction of two forms by estradiol,quantification in plasma and characterization in oocyte extract

Two forms of vitellogenin were isolated by DEAE agarose ion-exchange chromatography from plasma of the tilapia, Oreochromis mossambicus. The monomers have apparent molecular masses of 200 and 130 kDa, as indicated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and a total amount of phosphorus of 1.7 and 0.1%, respectively. Antibodies specific to the two forms, designated tVTG-200 and tVTG-130, were generated in rabbits and used to develop enzyme-linked immunosorbent assays (ELISAs) and in Western blot analyses of plasma and oocyte extract. SDS-PAGE of the oocyte extract showed a major protein band at 106.6, minor bands at 26.6, 24.2, and 23.7 kDa, and very faint bands at 83.4 and 17.5 kDa. Western blots of the oocyte extract revealed that the antiserum to tVTG-200 recognized strongly the protein bands at 24.2 and 23.7 kDa, and less strongly the bands at 25.1 and 22.6 kDa, whereas the antiserum to tVTG-130 recognized mainly the protein band at 106.6 kDa. The presence of both VTGs in untreated male tilapia was detected with the ELISAs using relatively high plasma volumes. Their presence in males was confirmed by VTG-like immunoreactive materials eluting from the ion-exchange column at the same positions as tVTG-200 and tVTG-130. The concentrations of the VTGs in males were several orders of magnitude lower than in vitellogenic females. Treatment of male tilapia with estradiol-17β (E₂) induced both VTGs within 24h. After 7 days, tVTG-130 reached a maximum concentration in plasma, whereas tVTG-200 continued to increase. Our findings demonstrate that the two vitellogenins are biochemically distinct, possibly differentially regulated, and made by both sexes.     

Comparative Oxygen Consumption and Metabolism of Striped Bass Morone saxatilis and its Hybrid M. chrysops♀ xM. saxatilis♂

It has been reported that metabolic rates of striped bass Morone saxatilis and hybrid striped bass M. chrysops♀ x M. saxatilis♂ are different. A series of experiments were conducted to further characterize oxygen consumption and metabolism of striped bass and its hybrid, the sunshine bass. Oxygen consumption was measured to determine standard and routine metabolic rates of striped bass and hybrid striped bass in a freshwater, flow‐through tank system. Additionally, blood chemistry stress indicators of the two bass groups were compared in both fresh and brackish water. Hematocrit (% PCV) and hemoglobin were measured in order to compare oxidative efficiencies of the bass. Plasma glucose, chlorides, and cortisol levels were measured to compare the relative stress status of the two bass types reared under experimental conditions. No significant differences were found in average daily oxygen consumption between striped bass and sunshine bass for either standard metabolism (P= 0.92), or routine metabolism (P = 0.86). Standard metabolic rates of oxygen consumption were 69 ± 4.1 and 68 ± 3.5 mg 0₂/kg³/⁴ bw/h for sunshine bass and striped bass respectively. Routine metabolic rates were 132 ± 30 and 125 ± 30 mg O₂/kg^3/4 bw/h for sunshine bass and striped bass respectively. While there were no significant differences in oxygen consumption between species, normal feeding activity generally resulted in increased oxygen consumption by the fish. Striped bass had significantly lower hematocrit values (P= 0.0001), but significantly higher hemoglobin concentrations than sunshine bass maintained in freshwater (P= 0.0001). Striped bass had significantly higher (P= 0.0001) levels of plasma glucose compared to sunshine bass (176 ± 8.6 vs. 103 ± 5.6 mg/dL respectively). Plasma chloride levels for striped bass (123 ± 1.9 mEq/L) were significantly higher (P= 0.041) than plasma chloride levels of sunshine bass (117 ± 1.7 mEq/L). Plasma cortisol levels were significantly higher (P= 0.0081) for striped bass (147 ± 8.4 ng/mL) compared to sunshine bass (119 ± 5.6 ng/ mL) when reared in freshwater. When maintained in brackish water, sunshine bass had significantly higher hematocrit values (P= 0.0001), and hemoglobin concentrations (P= 0.0012) when compared to striped bass. However, sunshine bass had significantly higher hemoglobin concentrations (P= 0.0012) when compared to striped bass. In addition, plasma glucose levels were significantly lower (P = 0.0079) for sunshine bass (79 ± 4.1 g/dL) when compared to striped bass (115 ± 11 g/dL). There were no significant differences between the bass in levels of chlorides or cortisol. No differences were detected in oxygen consumption. However, hybrid striped bass may have more efficient oxidative metabolism due to elevated hemoglobin concentrations. While striped bass hemoglobin values tended to be higher in brackish water than in freshwater, sunshine bass hematocrit or hemoglobin values generally were significantly higher than striped bass in both fresh and brackish water. Based on these results, hybrid striped bass may be capable of directing more energy towards growth than striped bass due to more efficient oxidative metabolism and lower losses of energy related to increased stress.     

Influence of temperature and salinity on length and yolk utilization of striped bass larvae

The effects of temperature and salinity on yolk utilization and growth of larval striped bass (Morone soxatilis) from Canadian maritime stocks were studied to determine optimal rearing conditions. Larval length increased during yolk utilization and maximum length (L_max) was attained at about 70 degree-days post-hatch. Dry weight declined during yolk utilization, whereas, larval wet weight was relatively constant and only declined when yolk was depleted. Temperature and salinity significantly affected the L_max. Temperatures exceeding 18 C resulted in lower L_max. Higher L_max values were attained at 5 % than at 1 or 10 %. Time to reach L_max was longer at 5 and 10 % than at 1 % for any experimental temperature. Time to reach L_max was 3 days post-hatch longer than times to maximum embryo dry weight. The Q₁₀ of yolk utilization was 1.71, 1.53 and 2.52 at 1, 5 and 10 %, respectively. Times to terminal yolk utilization were about 1.2 d shorter than times to L_max. Morphological changes during yolk utilization indicated rapid development of locomotory and predatory capabilities. These and other developmental strategies were compared to those of salmonids. Rearing striped bass larvae at 14 \-16C and 5 % throughout yolk utilization should result in longer larvae at initial first feeding.     

Physiology of seawater acclimation in the striped bass,Morone saxatilis (Walbaum)

Several experiments were performed to investigate the physiology of seawater acclimation in the striped bass, Morone saxatilis. Transfer of fish from fresh water (FW) to seawater (SW; 31–32 ppt) induced only a minimal disturbance of osmotic homeostasis. Ambient salinity did not affect plasma thyroxine, but plasma cortisol remained elevated for 24h after SW transfer. Gill and opercular membrane chloride cell density and Na⁺,K⁺-ATPase activity were relatively high and unaffected by salinity. Average chloride cell size, however, was slightly increased (16%) in SW-acclimated fish. Gill succinate dehydrogenase activity was higher in SW-acclimated fish than in FW fish. Kidney Na⁺, K⁺-ATPase activity was slightly lower (16%) in SW fish than in FW fish. Posterior intestinal Na⁺,K⁺-ATPase activity and water transport capacity (J_v) did not change upon SW transfer, whereas middle intestinal Na⁺,K⁺-ATPase activity increased 35% after transfer and was correlated with an increase in J_v (110%). As salinity induced only minor changes in the osmoregulatory organs examined, it is proposed that the intrinsic euryhalinity of the striped bass may be related to a high degree of “preparedness” for hypoosmoregulation that is uncommon among teleosts studied to data.     

The reproductive physiology of three age classes of adult female diploid and triploid brook trout (Salvelinus fontinalis)

Triploid female fish show impaired gametogenesis and are unable to produce viable offspring. The reproductive physiology of artificially-induced triploid female salmonids has been well described up until the time of first sexual maturation in diploids, but few reports exist for older triploids. This study reports the influence of triploidy on growth, ovarian development and reproductive endocrinology among three age classes of female brook trout (Salvelinus fontinalis) in comparison to sibling diploids. Triploids were larger than diploids for most of the study period, but the difference was statistically significant only during maturation and spawning of 2⁺ diploids. Plasma estradiol-17 (E₂), testosterone (T) and vitellogenin (VTG) levels in triploids were generally lower than in diploids, and VTG was the only parameter to show seasonal fluctuations resembling those of diploids. Triploids showed significantly lower GSI and total oocyte number than diploids of similar age, and only half of all triploids sacrificed during the study (n=56) had developing oocytes in their ovaries. At age 3⁺, 13 of 19 triploid females had oocytes at various stages of development, including perinucleolar, yolk vesicle and yolk globule stages. In addition, three of these fish had collectively produced 72 mature stage oocytes. Thus, whereas diploid brook trout can produce mature oocytes as two-year-olds, triploids cannot do so until four years of age, with the number of mature oocytes being greatly reduced.     

Reproduction of striped bass, Morone saxatilis (Walbaum), broodstock: monitoring maturation and hormonal induction of spawning

Abstract Levels of gonadal steroid hormones were quantified in an adult striped bass, Morone saxatilis (Walbaum), broodstock during their gametogenic cycle. Blood plasma concentrations of Estradiol (E₂) and testosterone in females, or 11-ketotestosterone(11-KT) and testosterone (T) in males, were used as indicators of maturation. In both sexes, hormone levels were low in summer but increased significantly by late October to intermediate levels which were then maintained until late January. They then increased again rapidly to maximum pre-spawning values attained in late February or March, and subsequently decreased during the spawning period (April and May) with an increased incidence of spent fish with low hormone levels. The changes in blood hormone concentrations coincided with annual changes in photoperiod and water temperature that may be useful landmarks for maturation in captive broodstock. Mature females were implanted with pellets containing a dose of approximately 20 μg/kg body weight of [D-Ala⁶-Pro⁹-Net]-LHRH (GnRHa) in a matrix of cholesterol (CH) and cellulose. In April, they had not yet begun final oocyte maturation (FOM) and were too immature for conventional induction of spawning by injection with human chorionic gonadotropin (hCG). In early April, females given two 95% CH (slow hormone-release) GnRHa pellets (95/95) or females given one 80% CH (fast hormone-release) GnRHa pellet and one 95% CH GnRHa pellet (80/95) spawned within 13 days treatment (n= 4) with good egg fertility (76 ± 7% of total) and hatch rates (62 ± 15% of fertile). Females given dual fast-release GnRHa pellets (80/80) or control (Sham) pellets did not spawn or show evidence of increased oocyte diameter or development. In late April, four of six females given the 80/95 GnRHa pellet combination spawned within 9 days. Three fish produced fertile eggs (54 ± 18%). one spawned overripe eggs, and the remaining two increased oocyte diameter and maturation. Three corresponding controls did not spawn, and two of these showed clear signs of atresia within 11 days. In early May, some females were undergoing early FOM and were mature enough to be spawned by hCG injection. Three were given a single 80% CH GnRHa pellet and spawned within 6 days of treatment to produce fertile eggs (44 ± 6%). Of two other females given dual 80% CH GnRHa pellets, one spawned infertile eggs and the other failed to spawn within 9 days. GnRHa implants show promise as a technique for inducing spawning of captive striped bass broodstock although the optimum hormone delivery systems, dosages and release rates should be verified for fish at specific maturational stages.