Survival and Growth of Bighead Carp Fry Exposed to Low Salinities

Bighead carp (Aristichthys nobilis Oshima) fry of various ages (11, 18, and 35 days post-hatch) were exposed to the low salinities encountered during the annual intrusion of seawater in Laguna Lake, Philippines. Practical indices of salinity tolerance assessed the effect of a 96 h direct exposure to low salinities (0–16). Mean (MST) and median survival times (MST50) of fry decreased as salinity of rearing medium increased. Younger fry were less able to tolerate exposure to these salinities than their older cohorts. Median lethal salinity after 96 h (MLS) revealed higher tolerance among 35–day old fry (7.6) than 11 (2.3) and 18–day old fry (6.0), demonstrating that survival in saline water depends on their age at initial exposure to low salinities. Mean body weight of 18–day old fry reared in 0 and 2 for 3 and 4 weeks was higher than for those reared in 4 and 6 for the same period. Growth over these periods was inversely related with the range of salinities tested. These results demonstrate that, despite their known stenohalinity, bighead carp fry possess some degree of osmoregulatory capability, allowing them to survive and grow in lakes subjected periodically to saltwater inflow.     

Brackishwater carp culture in potentially waterlogged areas using animal wastes as pond fertilizers

In order to assess the possibilities of utilizing drainage effluents (salinity range 5.0–12.5), fish culture experiments were carried out. Experiments on polyculture using cow dung (24 000 kg ha^–1 y^–1) as pond fertilizer were conducted at five different salinity levels (0.3–8.5). Studies have revealed that carp perform well in salinities up to 7.5 and reasonably high fish production has been obtained. Even though the ponds had a high trophic status, higher salinities ( > 7.5) appear to repress fish growth probably due to low dissolved oxygen (DO), high BOD and high NH₄-N. Experiments on monoculture of common carp (Cyprinus carpio) conducted at two different salinity levels (0.3–0.9 and 6.0–7.0) using four different organic fertilizers (cow dung at 24 000 kg and 20 000 kg, poultry at 1500 kg, duck at 6000 kg and sheep/goat at 1500 kg ha^–1 y^–1) have revealed the highest fish growth to be in poultry-treated ponds, followed in decreasing order by duck and sheep/goat wastes. Similar trends in fish production were observed both in fresh- and salt-water ponds. However, fish production was lower in ponds having higher salinities ( > 7.5). Nevertheless, these studies indicated that inland saline waters can be utilized for fish culture. With minor modifications in the existing technology of fish culture in stagnant freshwater fish ponds, animal wastes could be used to fertilize brackishwater fish ponds.     

Progressive transfer to seawater enhances intestinal and branchial Na+-K+-ATPase activity in non-anadromous rainbow trout

Two sizes of rainbow trout [170 (4), 91 (3) g] were progressively transferred to seawater in January 1995 using three steps of 9, 20 and 28. Na+-K+-ATPase activity in gills, intestine and kidney, and plasma sodium and magnesium levels were assessed in response to changed external salinity. Gradual transfer to seawater had a stimulatory effect on gill and intestinal Na+-K+-ATPase activities after the transfer to 28, while its activity remained unchanged in the kidney. Plasma sodium content was not modified, while magnesium levels increased in response to increased external salinity. The size-dependent response to seawater transfer described by other authors was not detected in our experiments. The results are discussed in terms of long-term adaptation to seawater.     

Annual cycle of plasma insulin and glucose of sea bass.Dicentrarchus labrax,L.

Annual plasma insulin and glucose cycles were studied inDicentrarchus labrax maintained in either seawater (37.8) or brackish water (3.5). In both media, the highest insulin levels were found during the prespawning period (August–November) coincident to increases in weight and a decrease in plasma glucose. During spawning (December–April) and postspawning (May–July) periods, the decrease in insulin occurred at the same time as a reduction in growth and an increase of plasma glucose. Temperature and salinity conditions impeded spawning in the brackish water group, in which a minor weight loss was regained more quickly than in the sea water group; insulin levels were also higher.     

Effects of acute temperature decreases on turbot fry and juveniles

Turbot fry (10–20 mm) and juveniles (85–110 mm) were transferred directly from 16.0–16.5 C to 1.0 C, 2.5 C, 5.5 C or 8.0 C seawater. The fry were more sensitive to cold water than juveniles. The fry survived for 1 week at 8.0 C but not at 5.5 C, whereas juveniles survived at 5.5 C but not at 2.5 C. Transfer of juveniles to 1.0 C and 2.5 C seawater caused a high mortality, a marked increase in plasma Cl^- concentration, decrease in muscle water content, and hyperglycaemia. Acclimation to 5.5 C (juveniles) or 8.0 C (fry and juveniles) markedly reduced the sensitivity to 1.0 C exposure.     

Studies on the use of hydrogen peroxide as a method for the control of sea lice on Atlantic salmon

Atlantic salmon (Salmo salar) post-smolts exposed to 1.23 hydrogen peroxide for 20 min at 13.5 C suffered an acute toxicity resulting in a 35% mortality within 2 h. Under similar conditions at 10 C no mortalities were observed with Atlantic salmon or goldsinny wrasse (Ctenolabrus rupestris). No histological changes were noted in tissues from exposed fish. Thirty-three per cent of adult and pre-adult sea lice (Lepeophtheirus salmonis) were immobilized or killed following exposure to 0.5 hydrogen peroxide at 10 C, rising to 98% at 2. Some lice were able to recover and regained normal swimming movements. Gas bubbles within the haemolymph caused affected lice to float on the water surface. A delay in the toxicity of hydrogen peroxide to copepodites occurred, with a 10% mortality following a 20 min exposure to 1.25 at 10 C rising to 100% mortality at 19 h post treatment.Dilute hydrogen peroxide was stable over the 20 min treatment period. Aeration and higher temperatures increased the long-term breakdown of a working concentration of hydrogen peroxide in seawater.     

Presence of maturation-promoting factor in 17α,20β-dihydroxy-4-pregnen-3-one-induced oocytes of catfish,Clarias batrachus

Full-grown immature Clarias batrachus oocytes respond in vitro to exogenous 17,20-dihydroxy-4-preg-nen-3-one ( 17,20-DP) by undergoing germinal vesicle breakdown (GVBD). Cytosolic extract (CE) prepared from 17,20-DP-induced oocytes has been shown to produce similar effect when microinjected into unstimulated immature oocytes of the same fish. A dose of 50 nl is enough to cause 100% GVBD after 4 h. Maturation-promoting factor was investigated from 17,20-DP-induced, immature and cycloheximide treated oocytes incubated in presence of [³⁵S] methionine. When the proteins were extracted and analyzed on SDS-PAGE, two prominent bands corresponding to molecular weight 34- and 46-kDa were detected in the CE of mature oocytes. However, labelling of [³⁵S] methionine was observed mainly in the region of 46 kDa protein band indicating de novo synthesis of this particular protein during l7,20-DP-induction. Further, immunoblotting study by using rabbit anti-cyclin B₁ antibody has clearly demonstrated that the protein which is newly synthesized is highly homologous to Xenopus cyclin B₁ and goldfish cyclin B.     

Seasonal,reproductive, and nutritional influences on muscle “buffering capacity” in yellow perch (Perca flavescens)

Effective non-bicarbonate buffering capacity (or buffer value) was measured in white muscle of yellow perch (Perca flavescens) by titrations with mineral acid and base in a carbon-dioxide free, closed system. Yellow perch were collected at three month intervals throughout 1983 from an acidic lake (pH 4.6) and two alkaline lakes (pH 7.8) in northern Wisconsin. Buffering capacity was also determined for white muscle of perch kept in the laboratory under different regimes of temperature and ration. The mean buffering capacity of white muscle from yellow perch taken directly from natural environments ranged from 40.7 ± 3.1 (SD) slykes in March of 1983 to 53.7 ± 2.8 (SD) slykes in July of that year. These changes in buffering capacity were strongly correlated with water temperature. Egg production and thirty-day laboratory starvation produced significant decreases in buffering capacity and increases in the water content of yellow perch muscle. Fed perch in the laboratory had a temperature dependent buffering capacity similar to field caught fish. Buffering capacity of white muscle did not differ between yellow perch from acidic and alkaline lakes. Investigators using buffering capacity as a gauge of species differences in metabolic potential, should be wary of seasonal and reproductive factors that might alter their conclusions.     

Protein kinase C in the spleen of the turbot (Scophthalmus maximus L.)

PKC activity was detected in spleen extracts from the turbot, Scophthalmus maximus, a teleost flatfish that is farmed commercially in several countries, in assays with the substrate EGF- R651–658 as phosphate acceptor. The activity was purified about 700-fold by a three-step chromatographic procedure (DEAE-cellulose, phenyl-Sepharose and threonine-Sepharose). Maximal activity was obtained in the presence of the typical PKC cofactors Ca2+ (0.1 mM) PtdS (20 g ml–1) and either DAG (2 g ml–1) or PMA (2 g ml–1). Activity was dose-dependently inhibited by H7 and by the PKC-specific inhibitors PKC19–36 and N-myristoylated PKC19–31. The rate of phosphorylation was highest with the PKC-specific substrate MARCKS161–175. In immunoblotting, MC5 (a mouse monoclonal antibody raised against bovine PKC) recognized bands of 80 and 100 kDa. Immunoblotting with antibodies raised against mouse PKC isozymes (, , , , , , and ) indicated the presence of all these isozymes in turbot spleen.     

Physiological and biochemical parameters for egg quality determination in lake trout,Salmo trutta lacustris

The present study investigated the relationships between egg viability and ovarian fluid composition, egg physiology and egg metabolism in lake trout, Salmo trutta lacustris, to obtain biomarkers for egg quality determination. The ovarian fluid pH, protein levels and activities of aspartate aminotransferase and -d-glucuronidase were significantly correlated with egg viability expressed as the number of eyed stage embryos. Regression models demonstrated that an ovarian fluid pH between 8.44 and 8.57, protein levels below 235.56 mg 100 ml^–1ovarian fluid, aspartate aminotransferase activity below 31.65 m min^–1 l^–1ovarian fluid and -d-glucuronidase activity below 8.62 m min^–1 l^–1 ovarian fluid characterized egg batches with high viability (80%).The increase in the egg wet weight during water hardening was also significantly correlated with the number of eyed stage embryos, and egg batches with high egg viability (80%) increased in wet weight by 13% during water hardening.From the investigated metabolic parameters the number of eyed stage embryos was significantly correlated with activities of NADP-dependent isocitrate dehydrogenase (egg viability 80% at 2.07 nM min^–1 mg^–1 protein) and NAD-dependent malate dehydrogenase (egg viability 80% at 47.25 nM min^–1 mg^–1 protein), with the respiration rate (egg viability 80% at 8.71 nM min^–1 mg^–1 protein), with the ratio of NADH to NAD levels (egg viability 80% 0.872), with the levels of free, non-esterified fatty acids (egg viability 80% 72.34 g mg^–1 protein), and the ratio of non esterified to esterified fatty acids (egg viability 80% at 0.749). Also, subjective and visual control methods were described to distinguish between batches with viable and non viable eggs.     

Influence of growth hormone on the hepatic mixed function oxidase and transferase systems of rainbow trout

The effect of GH treatment on hepatic cytochrome P450 content, aryl hydrocarbon hydroxylase (AHH), aminopyrine-N-demethylase (AND), testosterone hydroxylase, testosterone 5- and 5-reductase, UDP-glucuronyl transferase (UDPGT) and glutathione S-transferase (GST) activities in immature rainbow trout were investigated. Hepatic cytochrome P450 content, AHH and GST activities were measured in both GH implanted and GH injected animals whereas other activities were assayed in GH implanted trout only.GH implants significantly decreased cytochrome P450 content at 15 days compared to the control but no significant effect was observed at 15 or 30 d when GH was injected biweekly. In both cases, AHH activity was significantly decreased by GH treatment compared to the control whereas GST remained unchanged. Compared to the control, GH implanted fish exhibited a pronounced inhibition of AND, a decreased 6 and 16-testosterone hydroxylation, an inhibition of UDPGT with testosterone as substrate and an enhanced 17-testosterone oxidation.     

Steroidogenesis by ovaries and testes of the European catfish,the wels (Silurus glanis), in vitro

Testosterone, 3,17-dihydroxy-5-pregnen-20-one, 17,20-dihydroxy-4-pregnen-3-one (17,20P) and 5-pregnane-3,17,20-triol were identified as the major metabolites of [³H] 17-hydroxyprogesterone in ovarian incubations of the European catfish Silurus glanis. 17,20P and the reduced triol were present only in ovaries from fish primed with carp hypophysial homogenate (chh) while testosterone yields were significantly higher in controls than in treated fish. 11-Ketotestosterone, 11-hydroxytestosterone and 17,20-dihydroxy-4-pregnen-3-one (17,20P) were identified as the major metabolites of [³H]17-hydroxyprogesterone in in vitro incubations of testes of a spermiating catfish. There was no significant production of conjugates or other water soluble metabolites by either sex. The stimulation of plasma 17,20P, 17,20P and 11-hydroxytestosterone by chh in primed but not control males suggests that the role of these steroids in spermiation should be further examined.     

Distinct alpha (α)-subunits of salmon glycoprotein hormones: production sites in the pituitary with sexual maturity

Salmon pituitary glands contain two structurally distinct -subunit proteins (1 and 2) of glycoprotein hormones: the 2-subunit is common to all salmon gonadotropins (GTH I and GTH II), whereas the 1-subunit is present in only some GTH I molecules. GTH I is predominant in the pituitary gland and plasma during gametogenesis of salmon, but the roles of the 2 GTHs in gametogenesis remain unclear. To understand the roles of GTH I, it is important to clarify patterns of 1- and 2-subunit production with sexual maturity. Thus, we produced antisera that recognized the 1- or 2-subunit, and then immunohistochemically examined the production sites of these subunits in the trout pituitary gland during ovarian development. In all pituitary glands examined, the immunoreactivity of both the 1- and 2-subunits was strong in the GTH II-producing cells, although salmon GTH II, both 1- and II-subunits, has not been detected. However, GTH I-producing cells showed a less dense immunoreactivity for 1- and 2-subunits, whereas the I-subunit was abundant. On the other hand, TSH cells, reacted with 2 but not with 1.     

Gonadotrophin-releasing hormone agonist raises plasma concentrations of progestogens and enhances milt fluidity in male Atlantic halibut (Hippoglossus hippoglossus)

In two separate spawning seasons, spermiating male Atlantic halibut were implanted with pellets containing gonadotrophin-releasing hormone agonist (GnRHa). Males were bled repeatedly, and milt samples were collected. Blood samples were assayed for free and conjugated steroids: testosterone, 11-ketotestosterone, 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,20,21-trihydroxy-4-pregnen-3-one and steroids with a 17,20 configuration. Towards the end of the first season, pellets were implanted into three wild-caught and three hatchery-reared males. No control fish were available. The major progestogen in plasma was identified as sulphated 5-pregnane-3,17,20-triol (3,17,20-P-5-S). Concentrations of this steroid were stimulated by the GnRHa. Sulphated 17,20-P was also identified in the plasma, but at 10-fold lower concentrations than 3,17,20-P-5-S. In the middle of the second season, pellets were implanted into five hatchery-reared males; five unimplanted males were used as controls. Levels of androgens fell following GnRHa treatment, levels of progestogens rose briefly, and there was a significant increase in the fluidity of the milt. Of all the measured steroids, free and sulphated 17,20-P showed the best correlation with milt fluidity.     

Synthesis and regulation of 17α-hydroxy-20β-dihydroprogesterone in immature males of Oncorhynchus mykiss

Three experimental approaches were chosen to study the question if the progestin 17-hydroxy-20-dihydroprogesterone (1720OHP) is synthesised in testes of young Oncorhynchus mykiss, in which the absence of spermatozoa was verified histologically: first, in order to detect 20-hydroxysteroid dehydrogenase activity (20HSD), testes homogenates were incubated with ³H-labeled 17OHP.Metabolites were analysed by TLC, HPLC, and repeated crystallization to constant isotope ratios. One of the metabolites was identified as 1720OHP-³H, indicating that already immature testes contain 20HSD activity and are able to produce 20-reduced steroids. Second, 1720OHP was quantified by radioimmunoassay in incubates of testes fragments. The sensitivity of the gonads to gonadotropin II (GtH II) became evident when comparing incubations in the absence and presence of GtH II. Third, plasma levels of 1720OHP were significantly higher in animals injected with partially purified salmon gonadotropin, compared to controls. Thus, for the first time, it could be shown that 20HSD is present in testicular cells other than spermatozoa. Furthermore, 1720OHP is indeed secreted at a very early stage of testicular development; 1720OHP secretion is also responsive to GtH II. Future studies will have to show if the functions of this progestin include the stimulation of spermatogenesis.     

Progestogen metabolism by ovaries of the roach (Rutilus rutilus L.) and the rudd (Scardinius erythrophthalmus L.)

Roach ovaries converted 17-hydroxyprogesterone to 17,20-dihydroxy-4-pregnen-3-one (17,20P) and to glucuronides of testosterone and 17,20P. Small amounts of 5-pregnane-3- and -3, 17, 20-triols, 7-hydroxy-5-reduced metabolites and 17,20-dihydroxy-4-pregnen-3-one (17,20P) were also formed. Rudd ovaries converted this substrate mainly to 17,20P, 5-pregnane-3- and -3,17,20-triols, 17,20-dihydroxy-5-pregnan-3-one and testosterone glucuronide. The main metabolites of progesterone with both species were 17,20P, 5-pregnane-3,17,20-triol and 7-hydroxy-5-reduced steroids. Rudd ovaries formed, in addition, 17,20-dihydroxy-5-pregnan-3-one from progesterone. The pattern of metabolites was markedly altered when the concentration of substrate was increased from 42ng to 1 µg or 100 µg. At the highest concentration, glucuronides and polar steroids were not detectable, while at low concentrations they accounted for over 50% of the metabolites. 20-Hydroxysteroid dehydrogenase was shown to have a very high capacity, producing 21–47 µg 17,20P from 100 µg 17-hydroxyprogesterone substrate with 200 mg ovarian tissue in 5h.     

Relativein vitro effectiveness of estradiol-17β, androgens,corticosteroids, progesterone and other pregnene derivatives on germinal vesicle breakdown in oocytes of Indian major carps,Labeo rohita,Cirrhinus mrigala andCatla catla

The relative effectiveness of estradiol-17, androgens, corticosteroids, progesterone and other pregnene derivatives on germinal vesicle breakdown (GVBD) was investigatedin vitro using folliculated oocytes of three carps,Labeo rohita, Cirrhinus mrigala, andCatla catla. In all three species progesterone and 17-hydroxyprogesterone could induce GVBD but relatively 17,20-dihydroxyprogesterone was consistently found to be the most potent maturation-inducing steroid. Both estradiol-17 and testosterone were ineffective in inducing GVBD. Androsterone and dehydroepiandrosterone were found to be effective inC. catla at all the concentrations used. Deoxycorticosterone (DOC), hydrocortisone (HC) and cortisone were effective inducer of GVBD inC. catla whereas inL. rohita andC. mrigala only cortisone was found to be effective. All 5-reduced pregnenes were effective in inducing GVBD inL. rohita but inC. catla, only 5-pregnane-17-01-3,20-dione and 5-pregnane-3,17,20-triol and inC. mrigala, 5-pregnane-3-ol-20- one could induce oocyte maturation.     

Sulfation and uptake of the maturation-inducing steroid, 17α,20β-dihydroxy-4-pregnen-3-one by rainbow trout ovarian follicles

Rainbow trout ovarian follicles were incubated in vitro with tritiated 17,20-dihydroxy-4-pregnen-3-one (17,20-P; maturation-inducing steroid). Within 18–24 h, 56–66% had been converted to tritiated 17,20-dihydroxy-4-pregnen-3-one 20-sulfate (identification confirmed by HPLC) and 27% had been taken up (absorbed) by the follicles. Addition of 125 ng of cold (non-tritiated) 17,20-P to the incubations caused a decrease in the percentage of [³H]-17,20-P which was sulfated (56% 10%) and an increase in the percentage that was taken up (27% 57%). Seven steroids were tested for their effectiveness in decreasing the sulfation and increasing the uptake of tritiated [³H]-17,20-P. The order of effectiveness was in both cases the same: 17,20-P > cortisol > 11-deoxycortisol > 17,20,21-trihydroxy-4-pregnen-3-one > 17-hydroxy-4-pregnene-3,20-dione > 17-estradiol > testosterone. This indicated that the processes of sulfation and uptake of [³H]-17,20-P were related to each other and led to the hypothesis that, when cold 17,20-P is added to the medium, it reduces the proportion of [³H]-17,20-P which is sulfated and thus allows more free [³H]-17,20-P to enter the ovarian follicles. This hypothesis was supported by the finding that each ovarian follicle had the capacity in vitro to sulfate only ca. 2 ng of [³H]-17,20-P per 18h but a capacity to take up > 500 ng per 18h.Gonadotropin I, Gonadotropin II, forskolin and phorbol-12-myristate-13-acetate (which all have an affect on steroid biosynthesis) did not affect the amount of 17,20-P which was sulfated. Sulfating activity was localized in the thecal cell layer of the follicle. The yolk fraction was shown to be responsible for absorbing the [³H]-17,20-P.     

Influence of temperature on the hatching of eggs of Diplectanum aequans,a parasite of sea bass

This study deals with the influence of temperature on the hatching of eggs of Diplectanum aequans, a branchial ectoparasite of sea bass (Dicentrarchus labrax). At temperatures of 20 °C, 25 C and 30 °C hatching takes place within a few days of laying, whereas at 15 C and 10 C it occurs respectively between the 7th and the 12th day and between the 11th and the 19th day. No hatching has been observed at 5 C. Development is not completely inhibited at 5 C as the eggs can, at least partially, go on maturing and will subsequently hatch as soon as higher temperatures are encountered.     

Induced final maturation and ovulation in a small anabantoid teleost, the dwarf gourami, Colisa lalia. I. The effects of gonadotrophic preparations, LHRHa, steroids and thyroid hormones

Colisa lalia (Ham.) has been used as a model for the development of techniques for induced spawning that are applicable to small teleosts where ovulation requires prolonged exposure to suitable breeding conditions.Human chorionic gonadotrophin (hCG; 4 IU per fish) induced ovulation within 24 hours, whereas homologous pituitary extracts were relatively ineffective. When administered in saline ( 20 g per fish per injection), des-Gly10,[D-Ala6]- LHRH N-ethylamide (LHRHa) was ineffective, but it stimulated ovulation in a proportion of fish when administered ( 1.6 g per fish) as an emulsion in Freund's incomplete adjuvant (FIA). Together, these results suggest that ovulation requires the synthesis as well as the secretion of gonadotrophin in C. lalia.Long-term treatment with thyroid hormones appeared to enhance the ovulatory response to LHRHa in FIA, possibly by effects on the ovary; whereas the various steroids tested were ineffective at the dosages used