The ovarian regulation of ovulation in teleost fish

Ovulation is the release of a mature oocyte from its follicle wall enclosure in the ovary. This process requires the separation of the oocyte from the granulosa layer, the rupture of the follicle wall and the active expulsion of the oocyte through the rupture site. Results of experiments on various vertebrates, including fish, have shown that the control of these processes may involve the cooperative action of a number of ovarian regulators including proteases, protease inhibitors, progestational steroids, eicosanoids, catecholamines and vasoactive peptides. We have used two teleost models, the brook trout (Salvelinus fontinalis) and the yellow perch (Perca flavescens) to study the mechanism and control of ovulation in fish. Using subtractive cDNA cloning, a family of ovarian and ovulation specific mRNAs (TOPs = trout ovulatory proteins) was isolated from the brook trout ovary. These mRNAs have not previously been observed in the ovary of any vertebrate species; however, the proteins they code for have significant sequence homology to a group of mammalian protease inhibitors called antileukoproteinases. These inhibitors have been isolated from several mammalian mucosal tissues and their function may be to protect the mucosal lining from nonspecific degradation by proteases released from infiltrating leukocytes. The ovarian proteins encoded by the TOP mRNAs have now been characterized by Western blotting using antibodies derived against recombinant TOPs. Given the similarity of TOPs to antileukoproteinases, one function of TOPs may be to regulate proteolysis at the time of ovulation. In yellow perch, the maturational steroid, 17,20-dihydroxy-4-pregnen-3-one (17,20-PG), stimulates both germinal vesicle breakdown and ovulation in vitro. The stimulation of ovulation can be blocked by indomethacin, a prostaglandin endoperoxide synthase inhibitor. Thus, it appears that 17,20-PG acts through the production of an eicosanoid that is most likely a primary prostaglandin. This hypothesis is further supported by the observations that (1) a direct correlation exists between indomethacin levels that block ovulation and those that block primary prostaglandin synthesis in the ovary; (2) ovulation can be restored in indomethacin-blocked incubates with primary prostaglandins; (3) PGF levels increase at the time of ovulation in incubations of yellow perch follicles stimulated with 17,20-PG; and (4) the stimulation of PGF by steroids in the ovary is specific for 17,20-PG. Finally, 17,20-PG-stimulated ovulation and follicular prostaglandin synthesis requires the close interaction of extrafollicular tissue and other follicle wall layers.     

半滑舌鳎卵巢发育的组织学和形态数量特征研究

采用组织学方法系统研究了人工养殖条件下半滑舌鳎亲鱼卵巢的组织发育周年变化特征.结果表明,卵母细胞发育可分为6个时相,卵巢发育分为6期.卵巢不同发育时期都由不同类型的卵母细胞组成,半滑舌鳎为非同步分批多次产卵类型.周年发育过程中,6月龄前的鱼卵巢发育处于第Ⅰ期;9～12月龄卵巢可发育至第Ⅱ期;12月龄半滑舌鳎性腺可发育达到Ⅲ期,并一直保持至24月龄.3龄雌鱼达到性成熟,卵巢可发育至第Ⅳ期;随着水温和光周期的调控,卵巢发育进入成熟期(Ⅴ期),发生水合现象是卵母细胞成熟并准备排卵的信号;产卵结束后卵巢退化至第Ⅵ期,排出的卵母细胞退化吸收,以Ⅲ期卵巢越冬后,卵巢退化至Ⅱ期并持续至下一个繁殖周期.人工养殖条件下,卵巢内存在卵母细胞闭锁现象.性腺周年发育过程中,性腺指数(GSI)在性腺达到成熟期时达到峰值,产卵结束后性腺快速退化,GSI值各月份差异显著(P<0.05),亲鱼性腺重量在卵巢发育成熟过程中一直处于生长状态.亲鱼肝脏指数(HSI)在产卵前7～8月份(性腺达到成熟期前30 d左右)达到最大,表明7～8月份是性腺进行卵子发育、卵黄能量储备的重要时期,亲鱼在产卵前通过积极摄食储备体内能量,肝脏在卵母细胞卵黄积累方面具有重要的生理作用.肥满度(CF)值在繁殖盛期10～11月份达到最大,表现出与GSI类似的变化趋势.     

The effect of short-term starvation on ovarian development and egg viability in rainbow trout (Salmo gairdneri)

Adult female rainbow trout (Salmo gairdneri) were fed a commercial trout production diet until approximately 40 days prior to spawning at which time the fish were divided in two groups. One group continued to be fed while the other group was starved until spawning. Egg size, egg number, ovary weight, and egg proximate composition were examined periodically starting 5 months before spawning. Muscle tissues of the fish were sampled at spawning to measure somatic depletion of proximate consituents in the two groups. Egg hatchability was determined by incubating test lots from each spawning female. No significant differences (P > 0.05) were found in egg hatchability between the fed and starved groups. Changes in proximate composition were observed in the eggs during the months prior to spawning, but no differences were detected between the eggs of the starved and the fed groups. Short-term starvation did not significantly alter the proximate composition of muscle tissue of the spawned female trout.     

Effects of accelerated photoperiod regimes on the reproductive cycle of the female rainbow trout: II Seasonal variations of plasma gonadotropins (GTH I and GTH II) levels correlated with ovarian follicle growth and egg size

Female rainbow trout were exposed over their second reproductive cycle to a simulated natural photoperiod (control group) and to two accelerated photoperiod regimes (S9 and S6 groups). Early spawning was achieved in both accelerated groups, coupled, however, with a reduction of mean egg size. To investigate this reduction of egg size, circulating levels of GTHs and two indices of ovarian growth (gonadosomatic index and follicle diameter) were regularly measured in association with histological studies of structural changes in ovarian follicles. Regardless of the photoperiod regime, the seasonal profile of plasma GTH I levels appeared to be multiphasic. The successive transient elevations in GTH I levels appeared to be connected with the initiation of ovarian growth and vitellogenesis and also, with the synchronization of the late stages of maturation and ovulation. In contrast, the seasonal profile of plasma GTH II levels was monophasic, with a single peak at ovulation, confirming that GTH II is not associated with ovarian growth but promotes gamete maturation and release. Our results demonstrate that the reduction of egg size cannot be due to a deficient secretion of GTH I in plasma, since GTH I levels were much higher during vitellogenesis in both accelerated groups, but rather to an alteration of ovarian follicle growth during the late stages of vitellogenesis. Finally, the early and middle stages of ovarian growth appeared to be photosensitive periods, whereas the late stages were less so, and appeared to be controlled rather by an endogenous biological clock synchronized by the photoperiod.     

The dynamics of vitellogenin sequestration into vitellogenic ovarian follicles of the rainbow trout,Salmo gairdneri

This study provides quantitative data on the dynamics of protein sequestration into vitellogenic follicles of the rainbow trout, Salmo gairdneri. The ovarian uptake of both radiolabelled vitellogenin (VTG) and bovine serum albumin (BSA) were investigated in a homogenous population of maturing vitellogenic females. Ten fish were injected with ³H. VTG directly into the bloodstream. Concomitantly, five of these fish received an equal amount of ¹⁴C.BSA. Twenty two hours after injection, of the tissues sampled, the greatest proportions of ³H. VTG were present in the blood (into which the radio labels were administered) and in the ovary (up to 28% and 46% of that originally present in the blood, respectively). VTG uptake was both selective, rates of uptake far exceeding that of the ¹⁴C.BSA, and rapid. ³H. VTG was sequestered at rates of between 35 to 390 ng.mm² follicle surface⁻¹.h⁻¹ in the different fish. The rates of VTG uptake into similarly sized follicles varied both between different sites within the ovary (by up to 30%) and also between the ovaries (by up to 38%) of an individual fish.     

Confirmation of ovarian homogeneity in post-vitellogenic cultured white sturgeon,Acipenser transmontanus

Assessing stage of oocyte maturity in female sturgeon by calculating oocyte polarization index (PI) is a necessary tool for both conservation propagation managers and caviar producers to know when to hormonally induce spawning. We tested the assumption that sampling ovarian follicles from one section of one ovary is sufficient for calculating an oocyte PI representative of oocyte maturity for an individual animal. Short-wavelength near-infrared spectroscopy (SW-NIR) scans were performed on three positions per ovary for five fish prior to caviar harvest. Samples of ovarian follicles were subsequently taken from the exact location of the SW-NIR scans for calculation of oocyte PI and follicle diameter. Oocyte PI was statistically different though not biologically relevant within an ovary and between ovaries in four of five fish. Follicle diameter was statistically different but not biologically relevant within an ovary in three of five fish. There were no differences in follicle diameter between ovaries. No statistical differences were observed between SW-NIR spectra collected at different locations within an ovary or between ovaries. These results emphasize the importance of utilizing both oocyte PI measurement and progesterone-induced oocyte maturation assays while deciding when to hormonally induce spawning in sturgeon females.     

Fecundity, egg size and total egg volume differences in 12 stocks of rainbow trout, Oncorhynchus mykiss Richardson

Abstract. Measurements of egg number, egg size and total egg volume in relation to post-stripped weight, made on the eggs stripped from 12 separately maintained farm stocks of 4-year-old rainbow trout. Oncorhynchus mykiss Richardson, revealed highly significant differences between populations when analysed by regression and covariance statistical techniques. All of the stocks showed increases in fecundity with increasing fish size, with 61% (coefficient of determination or r²) of the variation in fecundity of the 12 stocks explained by the common regression on parental fish size. Comparison of the regressions of the different stocks revealed that the rate of increase of fecundity with increasing fish weight was constant throughout the weight range (i.e. the regressions had similar slopes) although there were highly significant differences in elevation, with some stocks producing almost twice as many eggs as some of the others. By contrast, the relationship of egg size with fish weight was much more variable with three of the stocks showing no significant regression of egg size on fish weight and a further two stocks with data poorly fitted to the regressions. Of the remaining seven stocks only one showed a significant difference in slope of the regression with modest increases in fish weight being associated with large increases in egg size. Only four of the remaining stocks of fish showed significant differences in the elevation of their regressions, i.e. the egg sizes were significantly different after the potentially conflicting influence of fish size was partitioned using covariance techniques. Assessments of total egg volume produced far improved regressions on post-strip weight with 75% (r²) of the variation in the pooled data of the 12 stocks being described by the line of the common regression. In general, there appeared to be a “trade-off” between egg number and size with the most and least fecund fish producing somewhat smaller and larger eggs respectively. All 12 regressions of totai egg volume on fish weight showed a common slope. However, there were significant differences in elevation of the regressions, with some of the stocks of trout producing up to a 55% greater volume of eggs. There appeared to be no relationship between the differences in egg number, size or total egg volume and the type or geograpfiical location of the farms on which the stocks were held. Although it remains to be determined to what extent these differences in reproductive performance are Inheritable, it is recommended that these characteristics should be taken into account by hatcheries in tbe design of long-term broodstock selection programmes for rainbow trout.     

Angiotensins stimulate in vitro ovulation and contraction of brook trout (Salvelinus fontinalis) follicles

The effects of salmon angiotensin I (sAI) and human angiotensin II (hAII) on in vitro ovulation of preovulatory (preOV) brook trout (Salvelinus fontinalis) follicles were investigated. Both angiotensins increased levels of ovulation above that in controls after 12 hours of incubation. The increase was statistically significant in incubates with greater than 1 M hAII. The effects of the angiotensins on follicle contraction were also studied indirectly by measuring the decrease in weight of punctured follicles taken prior to germinal vesicle breakdown. At 1 M, both angiotensins significantly decreased the weight of punctured follicles after 16 hours of incubation. The angiotensin-stimulated decrease in weight was not blocked by indomethacin (10 g/ml), indicating that follicle contraction was not prostaglandin-dependent. The data indicate that angiotensins might be directly involved in brook trout ovulation and the stimulatory effects of angiotensins on ovulation may be attributed to their effects on follicle contraction.This work was supported by NIH grant #HD25924-02.     

Specific gene expression profiles are associated with follicular maturational competence acquisition in rainbow trout (Oncorhynchus mykiss)

The expression profiles of several mRNAs were characterized during follicular maturational competence (FMC) acquisition. Post-vitellogenic female rainbow trout (Oncorhynchus mykiss) were assayed in vitro for follicular maturational competence (FMC). Ovarian follicles were stimulated in vitro for 60 h at 12 °C with a range of concentrations of partially purified gonadotropin and the efficient concentration for 50% germinal vesicle breakdown was calculated and used as an indicator of follicular maturational competence. Before in vitro assay, ovarian tissue was sampled in order to quantify mRNA abundance in the ovarian follicle by real-time PCR. The mRNA expression of Luteinizing Hormone receptor (LH-r), Follicular Stimulating Hormone receptor (FSH-r), Insulin-like Growth Factor 1 (IGF1), Insulin-like Growth Factor 2 (IGF2), Insulin-like Growth Factor receptor 1a (IGF-r1a) and 20β-hydroxysteroid dehydrogenase (20β-HSD), that are putatively expressed in the preovulatory ovary, was thus studied in females of varying FMC.     

Indicators of reproductive performance in rainbow trout Oncorhynchus mykiss (Walbaum) selected for high and low responsiveness to stress

A number of indicators of reproductive performance were assessed in rainbow trout divergently selected on the basis of their plasma cortisol response to a standardized stressor. For both male and female rainbow trout Oncorhynchus mykiss (Walbaum), body weight was significantly related to stress responsiveness. High‐responding (HR) females were significantly heavier than low‐responding (LR) females overall, and HR males were significantly heavier than LR males overall. There was no significant difference in the specific growth rate (SGR) of HR and LR female fish during the experimental period, whereas in contrast the mean SGR of HR males was significantly greater than that of LR males. The divergent confinement‐induced levels of blood cortisol in HR and LR groups were stable for more than 1 year after selection. There was no significant difference in the extent to which confinement stress reduced blood oestradiol‐17β levels in HR and LR females, despite a large difference in relative blood cortisol levels. The onset and rate of ovulation was similar within the HR and LR groups. Differences in fecundity, relative fecundity and egg volume and weight were wholly attributable to the different mean body weights of the HR and LR fish. There was no difference between the sperm counts of HR and LR males. There was significantly higher mortality among eggs derived from HR parents overall between fertilization and hatch, but maximum mean mortality did not exceed 12%. Mean time to eyeing in the HR groups was 224 degree days, and in the LR groups was 244 degree days. Hatching commenced at 344 and 347 degree days in the HR and LR groups, respectively, and was complete within 412 and 416 degree days respectively. Overall, selection for stress responsiveness was associated with no significant benefits or costs in reproductive performance.     

Oral transmission as a route of infection for viral haemorrhagic septicaemia virus in rainbow trout, Oncorhynchus mykiss (Walbaum)

Surveys among wild marine fish have revealed occurrence of viral haemorrhagic septicaemia virus (VHSV) infections in a high number of diverse fish species. In marine aquaculture of rainbow trout, preying on invading wild fish might thus be a risk factor for introduction and adaptation of VHSV and subsequent disease outbreaks. Our objective was to determine whether an oral transmission route for VHSV in rainbow trout exists. Juvenile trout were infected through oral, waterborne and cohabitation transmission routes, using a recombinant virus strain harbouring Renilla luciferase as reporter gene. Viral replication in stomach and kidney tissue was detected through bioluminescence activity of luciferase and qRT-PCR. Replication was detected in both tissues, irrespective of transmission route. Replication patterns, however, differed among transmission routes. In trout infected through oral transmission, replication was detected in the stomach prior to kidney tissue. In trout infected through waterborne or cohabitation transmission, replication was detected in kidney prior to stomach or in both tissues simultaneously. We demonstrate the existence of an oral transmission route for VHSV in rainbow trout. This implies that preying on invading infected wild fish is a risk factor for introduction of VHSV into marine cultures of rainbow trout.     

Stress response of juvenile rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)to chemical cues released from stressed conspecifics

The objective of this study was to determine whether exposure of rainbow trout (Oncorhynchus mykiss) to water containing a stressed trout or skin extract from stressed and non-stressed trout would elicit a stress response in conspecifics. Juvenile rainbow trout were exposed for 1 hour to water containing a stressed fish, homogenized skin extracts from a non-stressed fish, skin extract from a stressed fish and water with none of these factors. The stress response was measured over a 24-h period (1, 6, 12, 24 h after exposure). Plasma cortisol levels increased at 12 h in fish exposed to water from a stressed fish and skin extract from a stressed fish. Plasma glucose and hepatic hsp70 levels were not affected by treatments. The results suggest that rainbow trout elicit a stress response when exposed to stress-related alarm cues released from conspecifics.     

Food interrelationships between brown trout, Salmo trutta L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in a small put-and-take stillwater fishery

Abstract. The food of brown trout, Salmo trutta L., and rainbow trout, Oncorhynchus mykiss (Walbaum). in a small stillwater put-and-take fishery in Surrey, England was examined by stomach content analysis of fish caught by angling between the months of April and September 1985. Overall, brown and rainbow trout tended to utilize different food sources. Mann-Whitney U-tests showed most major food items to be eaten in significantly different amounts, the most important exceptions being chironomid pupae and adult Diptera. In all months except April, rainbow trout utilized mainly midwater food, particularly Cladocera. Brown trout fed mainly on benthic food organisms and fish. Spearman rank correlations showed diets to be dissimilar during this period, with significant negative correlations in May and August. These results indicate an absence of interspecific competition for food, and imply spatial separation of brown and rainbow trout. In April both brown and rainbow trout fed extensively on the temporary bottom fauna, mainly trichopteran and megalopteran larvae, and diets were significantly positively correlated.     

Acute dermatitis in farmed trout: an emerging disease

A new skin condition, known as puffy skin disease (PSD), emerged in farmed rainbow trout Oncorhynchus mykiss (Walbaum) in 2002. The number of new cases increased considerably from 2006. Clinical signs include white or grey skin patches, which become raised and red with excessive mucous production and scale loss. Fish are inappetant and lose condition. Histologically, the key feature is epithelial hyperplasia. We undertook a questionnaire study of trout farmers in England and Wales to investigate prevalence and risk factors. PSD was reported on 37% (n = 49) of rainbow trout sites, located in 28 river catchments. The increase in cases from 2006 onwards was mirrored by the increase in red mark syndrome (RMS). Prevalence and severity of PSD were highest in the summer months. The presence of PSD was associated with RMS (OR = 9.7, P < 0.001). Sites receiving live rainbow trout in the previous 12 months were considerably more likely to have PSD (OR = 5.3. P < 0.01), which suggests an infectious aetiology. The size of affected fish and prevalence varied between farms, indicating that farm‐level factors are important. Future research should further investigate the aetiology of PSD and practices to manage the disease.     

Egg Quality Determinants in Finfish The Role of Overripening with Special Reference to the Timing of Stripping in the Atlantic Halibut Hippoglossus hippoglossus

Egg quality, those characteristics of the egg that determine its capacity to survive, is a significant problem for many species of fish currently being farmed. Little is known about the determinants of egg quality and there is little agreement regarding reliable methods for its assessment. To be of practical benefit, assessments should be simple to perform and should be carried out as early in development as is possible. Fertilization rates are often used as measures of quality. For the Atlantic halibut Hippoglossus hippoglossus, fertilization rate and assessments of cell symmetry at early cleavage stages provide reasonable indicators of quality. Regardless of assessment method, it is strongly recommended that performance data from all batches of each broodfish be examined when surveying the overall quality of a stock. The misleading effects of pooling such information are demonstrated for a rainbow trout Oncorhynchus mykiss broodstock. Although a large number of factors have been implicated as possible determinants of egg quality, only 1) bacterial colonization of the eggs, 2) nutritional status of the broodfish and, 3) overripening, the process of aging that occurs when eggs are retained within the broodfish after ovulation, have been clearly shown to affect egg quality. The effect of overripening on fish egg quality is discussed in detail. Species-specific differences in the time scale of overripening are pointed out and related to spawning strategy and water temperature. Rainbow trout eggs and those of other salmonids should be fertilized within approximately one week of ovulation. Overripening proceeds much more rapidly in warm water species, e.g., tilapia eggs must be fertilized within an hour or so of ovulation. Egg viability also decreases rapidly for batch spawning species. Fertilization data are presented for Atlantic halibut demonstrating that egg quality decreases 4–6 h after ovulation. The rate of overripening was comparable for eggs held in vitro in ovarian fluid to those retained within the ovarian lumen. These halibut data, combined with information from other marine and freshwater fish, indicate that overripening is a significant determinant of egg quality for many if not all fish.     

Comparative sensitivity of carp, Cyprinus carpio L. and rainbow trout, Salmo gairdneri Richardson, to Renibacterium salmoninarum

Abstract. The pathogenicity of Renibacterium salmoninarum to carp, Cyprinus carpio L., and rainbow trout, Salmo gairdneri Richardson, was investigated. All carp injected with 4·8 × 10⁸ cells/fish, or 4·8 × 10⁷ cells/fish survived for 38 days. R. salmoninarum was isolated from all moribund fish, but not from the kidney of surviving fish, although R. salmoninarum antigen was detected in several of these fish by the dot blot assay. On the other hand, mortality in rainbow trout was 95% in the fish injected with 4·8 × 10⁸ cells/fish, and 15% in those which received 4·8 × 10⁷ cells/fish. R. salmoninarum antigen was detected by the dot blot assay in all surviving rainbow trout. The number of R. salmoninarum cells was immediately decreased by carp or rainbow trout serum, and the serum bactericidal activity of carp was higher than that of rainbow trout. Carp blood leucocytes had higher phagocytic activity than those of rainbow trout.     

Variation in semen production of farmed Atlantic salmon and rainbow trout

The variation in semen production among farmed Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) has been studied. Both species were stripped at weekly intervals, the Atlantic salmon four times and the rainbow trout three times.The individual variation in volume of semen was very high, particularly in rainbow trout. The total volume of semen obtained was 137 ml (20 ml/kg body weight) in Atlantic salmon and 23 ml (5 ml/kg body weight) in rainbow trout. The intraclass correlation for volume of semen was estimated at 0.73 in Atlantic salmon and at 0.59 in rainbow trout. The correlations between volume of semen and body size (weight and length) were all positive. They were all significant and medium in Atlantic salmon whereas in rainbow trout they were all low and significant only for volume of semen at first stripping.The number of males needed to supply the Norwegian fish farming industry with semen is discussed. It is concluded that the possibility of disseminating genetic improvement throughout the whole population of farmed Atlantic salmon and rainbow trout by transport of semen from selected males is considerable.     

Quantifying risks of volitional consumption of New Zealand Mudsnails by Steelhead and Rainbow Trout

To assess the risk of transferring alien, invasive New Zealand mudsnails (NZMS) Potamopyrgus antipodarum with shipments of live rainbow or steelhead trout Oncorhynchus mykiss, we conducted laboratory trials to quantify and determine the volitional ingestion of snails from the bottom of laboratory tanks. Approximately, 2000 snails were placed on the bottom of a test tank and groups of 10 fish were added to each tank. After 48 h, the fish were removed, euthanized and the snails in the gastrointestinal tract were counted, and the proportion of snails remaining in each tank was measured. We found that both rainbow trout and steelhead consumed NZMS, but rainbow trout consumed nearly twice the number of snails that were consumed by steelhead trout. Feeding fish a maintenance diet increased the total consumption of snails by fish to an average of 64.5 snails per rainbow trout. Our study suggests that management strategies that depurate fish without feed for 48 h to reduce the risk of transfer of NZMS are not viable in preventing the transport of snails if rearing waters contain snails.     

Effects of Oral Administration of Specific Antibodies to Rainbow Trout Oncorhynchus mykiss

A new product called oralized fish serum concentrate (OFSC) was evaluated for a possible effect against various bacterial pathogens in rainbow trout. The OFSC produced from immune trout sera was found to contain fully functional antibodies and complement component C3. The antibodies detected in the serum concentrate were specific to Vibrio anguillarum (O1 and O2) and Aeromonas salmonicida , which had been used for vaccination of the fish prior to serum collection. The functionality of the specific antibodies in OFSC was not reduced after 6 wk storage at -20 C, 5 C, and 20 C. The serum was mixed with commercial trout feed and used for feeding rainbow trout fry (first feed period). After oral delivery of OFSC to rainbow trout for 1 mo, samples of gut content and gut tissue contained functional antibodies. In gutted fish no functional antibodies were found. This suggests that antibodies from OFSC are unable to be transferred across the gut wall in a functional state. Oral administration of OFSC did not increase survival of rainbow trout in an immersion challenge with Vibrio anguillarum .     

Evaluation of AQUI-STM (efficacy and minimum toxic concentration) as a fish anaesthetic/sedative for public aquaculture in the United States

A preliminary evaluation of efficacy and minimum toxic concentration of AQUI-S^TM, a fish anaesthetic/sedative, was determined in two size classes of six species of fish important to US public aquaculture (bluegill, channel catfish, lake trout, rainbow trout, walleye and yellow perch). In addition, efficacy and minimum toxic concentration were determined in juvenile–young adult (fish aged 1 year or older) rainbow trout acclimated to water at 7°C, 12°C and 17°C. Testing concentrations were based on determinations made with range-finding studies for both efficacy and minimum toxic concentration. Most of the tested juvenile–young adult fish species were induced in 3 min or less at a nominal AQUI-S^TM concentration of 20 mg L^–1. In juvenile–young adult fish, the minimum toxic concentration was at least 2.5 times the selected efficacious concentration. Three out of five species of fry–fingerlings (1.25–12.5 cm in length and < 1 year old) were induced in ≤ 4.1 min at a nominal concentration of 20 mg L^–1 AQUI-S^TM, with the other two species requiring nominal concentrations of 25 and 35 mg L^–1 for similar times of induction. Recovery times were ≤ 7.3 min for all species in the two size classes. In fry–fingerlings, the minimum toxic concentration was at least 1.4 times the selected efficacious concentration. There appeared to be little relationship between size of fish and concentrations or times to induction, recovery times and minimum toxic concentration. The times required for induction and for recovery were increased in rainbow trout as the acclimation temperature was reduced.