Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and ovulation rate in female coho salmon, Oncorhynchus kisutch, close to final maturation.

Plasma levels of 17beta-estradiol, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P), and testosterone were measured in adult female coho salmon in late vitellogenesis, approximately 1.5 months before spawning and just before and following intraperitoneal injection with the aromatase inhibitor (AI) Fadrozole. Injection at dosages of 0.1, 1.0, and 10.0 mg AI/kg body wt caused a significant drop in plasma 17beta-estradiol levels relative to preinjection values within 3 or 6 h. Injection of 10 mg AI/kg body wt caused a significant increase in plasma 17alpha-20beta-P levels within 3 h. Ten days after injection 67% of the fish treated with 10 mg AI/kg body wt had ovulated in contrast with 0% in the group injected with 0.1 mg AI/kg body wt. The fertilization rate of the eggs varied between 96% in the control group and 85% in the groups injected with AI. We conclude that the shift from 17beta-estradiol to 17alpha,20beta-P biosynthesis, which is characteristic of maturing Oncorhynchus sp., was advanced significantly by treatment with AI and that Fadrozole can be used as a tool to investigate periovulatory endocrine changes in salmon.

The effect of the aromatase inhibitor fadrozole and two polynuclear aromatic hydrocarbons on sex steroid secretion by ovarian follicles of coho salmon.

A variety of endogenous and exogenous factors can influence sex steroid production by salmon ovarian follicles and ultimately impact reproductive development. We examined the effect of an aromatase inhibitor, fadrozole, and common environmental contaminants (PAHs) on sex steroid secretion by ovarian follicles. Ovarian follicles of coho salmon were incubated in vitro with various concentrations of testosterone (0.10-0.40 microM) and fadrozole (10 and 100 microM), or with varying doses (between 0.05 and 5.0 microM) of the PAHs beta-naphthoflavone (BNF) and 20-methylcholanthrene (20-MC). 17 beta-Estradiol secretion was significantly reduced when follicles were incubated in the presence of fadrozole, BNF, or 20-MC. In contrast, 17 beta-estradiol production by ovarian follicles increased in a dose-dependent manner when incubated with increasing doses of the aromatizable androgen testosterone. Although increasing doses of PAHs significantly reduced follicular 17 beta-estradiol production no effect on testosterone secretion was observed. Hence, both fadrozole and PAHs can significantly reduce 17 beta-estradiol secretion by salmon ovarian follicles and may affect female sexual development.

Plasma steroid and corticosteroid levels in female pacu Piaractus mesopotamicus, Teleostei-Characidae.

We report the plasma levels of estradiol-17 beta (E2), testosterone (T), 17 alpha-20 beta-dihydroxy-4-pregnen-3-one (17-20P), and cortisol (F) in female pacu during the reproductive cycle (N = 44) and in females induced to ovulate with an analogue of luteinizing hormone releasing hormone (LHRHa; 10 micrograms/kg) (N = 24). The plasma hormone levels were determined by validated radioimmunoassays. Females sampled during the reproductive cycle were grouped into 4 gonadal stages: resting, early maturation, advanced maturation and regression. The calculated gonadosomatic index varied from 0.5 +/- 0.1% in resting stage to 8.1 +/- 0.6% in advanced maturation stage. The E2 and T values were highest during the early maturation stage (E2 = 2172 +/- 7.1 pg/ml; T = 412 +/- 58 pg/ml) and the F values were highest during the advanced maturation stage (132 +/- 5 ng/ml). Females induced to ovulate by LHRHa injection were sampled at 0.6, and 12 h after injection of LHRHa. Two additional groups were sampled at ovulation and 24 h after ovulation. The E2 values were highest at 6 h (2917 +/- 65 pg/ml). The T and F values were highest at ovulation (T = 3498 +/- 77 pg/ml; F = 387 +/- 16 ng/ml) and 17-20P was detected only at ovulation (2163 +/- 80 pg/ml).

Plasma steroid and corticosteroid levels in female pacu Piaractus mesopotamicus, Teleostei-Characidae

We report the plasma levels of estradiol-l7Beta (E2), testosterone (T), 17(alpha-2Obeta-dihydroxy-4-pregnen-3-one (l7-2OP), and cortisol (F) in female pacu during the reproductive cycle (N = 44) and in females induced to ovulate with an analogue of luteinizing hormone releasing hormone (LHRHa; 10 mug/kg) (N = 24). The plasma hormone levels were determined by validated radioimmunoassays. Females sampled during the reproductive cycle were grouped into 4 gonadal stages: resting, early maturation, advanced maturation and regression. The calculated gonadosomatic index varied from 0.5 ñ 0.1 per cent in resting stage to 8.1 ñ 0.6 per cent in advanced maturation stage. The E2 and T values were highest during the early maturation stage (E2 = 2172 ñ 7.1 pg/ml; T = 412 ñ 58 pg/ml) and the F values were highest during the advanced maturation stage (l32 ñ 5 ng/ml). Females induced to ovulate by LHRHa injection were sampled at 0, 6, and 12 h after injection of LHRHa. Two additional groups were sampled at ovulation and 24 h after ovulation. The E2 values were highest at 6 h (2917 + 65 pg/ml). The T and F values were highest at ovulation (T = 3498 + 77 pg/ml; F = 387 ñ 16 ng/ml) and 17-20P was detected only at vulation (2163 ñ 80 pg/ml).

Increases in gill cytosolic corticosteroid receptor abundance and saltwater tolerance in juvenile coho salmon (Oncorhynchus kisutch) treated with growth hormone and placental lactogen.

Juvenile coho salmon (Oncorhynchus kisutch) were injected with one of two recombinant bovine hormones, growth hormone (bGH; 5.0 and 0.5 micrograms.g-1 body wt) or placental lactogen (bPL; 5.0, 0.5, and 0.05 micrograms.g-1 body wt) to determine the effect on growth, plasma cortisol concentration, cytosolic corticosteroid receptors (CR) in the gills, and the development of hypoosmoregulatory ability. One week following a single injection or six weekly injections of bGH or bPL, the fish were measured and sampled for CR concentration and Na+,K(+)-ATPase activity in the gills. Fish were also challenged with salt water (salinity 25%) for 24 hr to determine saltwater tolerance at the end of the 6-week treatment. Treatment with bPL and bGH significantly increased weight and length of the fish. The 0.05-micrograms bPL dose significantly elevated plasma cortisol concentration, whereas all other hormone treatments did not affect cortisol levels. bPL and bGH also significantly increased CR concentration and Na+,K(+)-ATPase activity in the gills. The perturbation in plasma sodium concentration was least in animals receiving the highest dose of bPL and the bGH-treated animals following transfer to seawater. An increase in cytosolic CR by bGH and bPL may increase responsiveness of the gills to cortisol and partially account for the increase in Na+,K(+)-ATPase activity and greater ability to regulate plasma sodium in seawater as exhibited by the experimental groups.

A radioimmunoassay for oncorhynchid growth hormone targeted to the physiological range.

This study describes the development of an oncorhynchid growth hormone (GH) radioimmunoassay using recombinant chum salmon GH (rsGH) and a rabbit antiserum (TJK-1) raised against this recombinant material. The assay was designed to measure the wide range of circulating immunoreactive GH (IRGH) levels in Pacific salmonids, resulting in a standard curve capable of accurately determining plasma levels of IRGH from 0.5 to 250 ng/mL without dilution. The assay ED50 and ED90 values averaged 13.1 and 0.5 ng/mL, respectively. This radioimmunoassay specifically recognizes oncorhynchid IRGH, showing no cross-reactivity with recombinant porcine and bovine GH, or natural chum salmon prolactin at concentrations up to 10 micrograms/mL. Curves approximately parallel to the standard curve were obtained with purified natural coho salmon GH and plasma from chinook salmon. Recovery of rsGH from plasma was complete over the full range of the standard curve. Intra- and inter-assay coefficients of variation were 6.0 and 12.9%, respectively. Plasma IRGH levels in fed coho salmon were 30.6 +/- 5.3 ng/mL, while those in fish starved for 2 weeks were 132.9 +/- 53.9 ng/mL. Starvation for an additional 4 weeks had no significant effect. Plasma IRGH levels in control rainbow trout injected with saline were significantly higher 45 min post-injection. In contrast, fish injected with recombinant porcine GH exhibited no elevation in IRGH. It is speculated that exogenous GH inhibits the production of endogenous GH.

Bovine placental lactogen is a potent stimulator of growth and displays strong binding to hepatic receptor sites of coho salmon.

Juvenile coho salmon were treated with bovine placental lactogen (bPL) and bovine growth hormone (bGH) to examine the growth promoting activities of these proteins in a lower vertebrate. Fish were intraperitoneally injected either with 0.5 or 5.0 micrograms/g bPL or with 5.0 micrograms/g bGH once a week for 5 weeks. After only a single injection and 1 week of growth, the high dose of bPL stimulated a significant increase in weight and length relative to untreated fish or fish treated with a control protein, bovine serum albumin. At the end of the experiment, all hormone-treated groups were significantly larger than controls. Fish treated with 5 micrograms/g bPL gained more than three times as much weight as controls. The 5.0 micrograms/g bGH group grew at the same rate as fish treated with one-tenth this dose of bPL, indicating that bPL is a potent stimulator of growth in this species. Radioreceptor assays performed on coho salmon liver membrane preparations indicate that bPL binds with approximately 430-fold higher affinity than bGH, and some 8000-fold higher affinity than bovine prolactin. The action of bPL relative to the structure and function of salmonid pituitary hormones is discussed.

Uptake and clearance of exogenous estradiol-17ß and testosterone during the early development of coho salmon (Oncorhynchus kisutch), including eggs, alevins and fry.

The uptake and clearance of estradiol-17ß (E2) and testosterone (T) were examined during the initial stages of development of coho salmon (Oncorhynchus kisutch), including eyed-eggs, newly hatched alevins and first feeding fry. Radiolabeled steroids were administered through the water in tracer amounts with or without their nonradioactive form at 400 µg l(-1). Regardless of developmental stage, saturation levels were invariably attained earlier for T than for E2, thus resulting in a higher incorporation of E2. However, both steroids had similar clearance patterns. Uptake and clearance was clearly stage-dependent, being fastest in fry, intermediate in alevins and slowest in eggs. Furthermore, combined uptake and clearance patterns showed that exposure to steroid was also higher for E2 than for T and stage-dependent, but always markedly highest in alevins. Subsequently, based on the observed elimination of the estrogen, a double immersion in E2 at 400 µg 1(-1), administered 2 days apart to maximize exposure during the alevin stage, was assayed for its effect on sex reversal and found to induce the production of 100% females. We suggest that the yolk, which is present in substantial amounts during the initial stages of development in salmonids, can retain the exogenously administered liposoluble steroids, thus providing developing embryos with an extended supply of, and exposure to, these steroids well after the treatment is finished. Together, these findings help to explain the previously observed high effectiveness of sex steroids administered during early development in regulating gonadal differentiation in salmonids, the higher effectiveness of E2 compared to T, and clarify the localization of the most sensitive period to the action of exogenous steroids at the alevin stage in the coho salmon.

Antisomatostatin-induced growth acceleration in chinook salmon (Oncorhynchus tshawytscha).

Since somatostatin (SRIF) inhibits the release of growth hormone (GH), its immunoneutralization may provide an alternative to GH therapy as a means of enhancing somatic growth in fish. The present study examined the feasibility of accelerating growth in juvenile chinook salmon by means of antiSRIF administration. Yearling salmon of Nicola River stock (BC, Canada) were injected intraperitoneally every 5 days, for a total of 40 days, with either SRIF (1 µg g-1 body wt.), antiSRIF (SOMA-10, 1 µg g(-1)), recombinant bovine GH (rbGH, 2.5 µg g(-1)), recombinant porcine GH (rpGH, 2.5 µg g(-1)) or saline (controls). No significant differences were observed in length, weight or final condition factor (k) between the SRIF-treated and control fish over the experimental period. However, the fish treated with the antiSRIF were significantly (p ≤ 0.05) longer and heavier than the control salmon after 25 and 30 days respectively. Furthermore, antiSRIF treatment caused a lowering in k when compared to the control salmon. Fish injected with rbGH or rpGH were significantly longer and heavier than all other groups (p ≤ 0.05), after only 5 days. GH treated groups also returned higher k when compared against all other treatments (p ≤ 0.05). No differences were observed in growth between the two rGH treatments over the experimental period.

Effects of natural, synthetic, aromatizable, and nonaromatizable androgens in inducing male sex differentiation in genotypic female chinook salmon (Oncorhynchus tshawytscha).

The relative potency of several androgens to induce the male phenotype in sexually undifferentiated genotypic female chinook salmon were compared in two separate experiments. The aromatizable and nonaromatizable androgens testosterone (T) and 11-ketotestosterone (11-KT), and the synthetic aromatizable and nonaromatizable androgens 17 alpha-methyltestosterone (MT) and 17 alpha-methyldihydrotestosterone (MDHT) were administered to newly hatched alevins in a single 2-hr immersion treatment at doses ranging from 3.2 micrograms/liter to 10 mg/liter. The influence of these treatments on sex differentiation was evaluated by the histological examination of the resulting gonads 6 and 11 months later. In the control group, which was not exposed to exogenous steroids, no males or intersex fish were observed. In contrast, essentially 100% masculinization occurred in groups exposed to MDHT at dosages of 400 micrograms/liter and higher. Treatment with the aromatizable androgen MT resulted in a dose-dependent masculinization, with the production of 100% males at 400 micrograms/liter. However, higher doses resulted in fewer males. 11-KT and T were less potent than the synthetic androgens. The number of males produced after treatment with 11-KT followed a dose-dependent pattern while T showed virtually no masculinizing effect in inducing male phenotype in these studies. The resultant AD50 dosage levels (dosage at which 50% of the genotypic females were sex-reversed into phenotypic males) after a single 2-hr immersion treatment were: 30, 60, and 500 micrograms/liter for MDHT, MT, and 11-KT, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth enhancement following dietary delivery of recombinant porcine somatotropin to diploid and triploid coho salmon (Oncorhynchus kisutch).

Diploid (2n) and triploid (3n) coho salmon were fed upon control (diet 1), recombinant porcine somatotropin (rpST)-supplemented (diet 2; 20 µg rpST/g body wt/day) or rpST/antacid/detergent-containing feed (diet 3; 20, 100 & 20 µg/g body wt/day respectively) for 16 wk. Fish were weighed and measured bimonthly and their relative performances assessed. By wk 4, 2n and 3n groups fed upon diet 2 were significantly heavier and longer than control 2n fish. At the termination of the trial, diet 3 fed animals were greater in weight than all other treatment groups. Diet 3 salmon also returned better feed conversion efficiencies than either diet 1 or 2 groups. RpST therapy induced a 28.7% and 60.2% increase in group wt for 3n diet 2/3 coho versus (-vs-) controls. Likewise, 2n rpST-treated fish increased wt -vs-2n control coho by 17% and 50% respectively over the same period. No differences were recorded between groups for body moisture, but diet 2, 3n and both diet 3 groups exhibited decreased condition factors when compared to control fish (p ≤ 0.05).

Dosage-dependent differences in the effect of aromatizable and nonaromatizable androgens on the resulting phenotype of coho salmon (Oncorhynchus kisutch).

This study was conducted to investigate whether aromatization to estrogen could be the cause for the paradoxical feminization of gonads of sexually-undifferentiated fish after treatment with androgen at either high doses or for long periods. The aromatizable androgen 17α-methyltestosterone (MT) and the nonaromatizable androgen 17α-methyldihydrotestosterone (MDHT) were administered to groups of newly hatched coho salmon (Oncorhynchus kisutch) in a single 2h immersion at concentrations ranging from 6.25 to 6,400µg/l. The effects of treatment were evaluated by determining the resultant proportion of males in each experimental group. The effects of steroid administration on the final mean weight, length and condition factor were also determined. An increase in all these three variables was observed in the groups treated with the higher doses of MT. Regarding the resultant sexual phenotype, the response to both androgens was similar at the majority of doses tested. However, at the highest dose, the proportion of females increased with respect to that of males for MT, but not for MDHT. Since the major difference between the two androgens tested is their capacity to be aromatized, it seems that aromatization to estrogen, rather than inhibition of the biosynthesis of endogenous androgen, may explain the paradoxical feminization encountered.

Toward the origin of the secondary palate. A possible homologue in the embryo of fish, Onchorhynchus kisutch, with description of changes in the basement membrane area.

The oral cavity of embryos and larvae of the teleost Onchorhynchus kisutch was examined. Tissues were obtained at different ages prior to and after hatching and processed for transmission and scanning electron microscopy. A bilaterally symmetrical bulge developed from the superolateral aspect of the oral cavity and projected toward its floor, along the sides of the tongue. The bulge extended from behind the primary palate to a position midway below the eye, anterior to the gill arches, and it is suggested to be the homologue of the secondary palate of higher vertebrates. Ultrastructurally, the epithelium differentiated as the stratified squamous type and it contained mucous cells. However, the features of programmed cell death seen during palatogenesis in mammals were absent in fish. The fish palate mesenchyme, unlike that of higher vertebrates, was chondrified. Also in contrast to higher vertebrates, alterations were seen in the fish palatal basement membrane. A transient appearance of adepidermal granules in the lamina lucida region was followed by organization of collagen fibrils, first into an orthogonal pattern and then into a herring-bone arrangement, in the lamina reticularis region. There was no further advancement in the morphogenesis of fish palate. It is suggested that the differences in the morphogenesis and structure of the secondary palates of various vertebrates may reflect environmentally enforced adaptation, resulting in different programming of cells.

Determination of antimony in ores and related materials by continuous hydride-generation atomic-absorption spectrometry after separation by xanthate extraction.

A continuous hydride-generation atomic-absorption spectrometric method for determining approximately 0.02 mug/g or more of antimony in ores, concentrates, rocks, soils and sediments is described. The method involves the reduction of antimony(V) to antimony(III) by heating with hypophosphorous acid in a 4.5M hydrochloric acid-tartaric acid medium and its separation by filtration, if necessary, from any elemental arsenic, selenium and tellurium produced during the reduction step. Antimony is subsequently separated from iron, lead, zinc, tin and various other elements by a single cyclohexane extraction of its xanthate complex from approximately 4.5M hydrochloric acid/0.2M sulphuric acid in the presence of ascorbic acid as a reluctant for iron(III). After the extract is washed, if necessary, with 10% hydrochloric acid-2% thiourea solution to remove co-extracted copper, followed by 4.5M hydrochloric acid to remove residual iron and other elements, antimony(III) in the extract is oxidized to antimony(V) with bromine solution in carbon tetrachloride and stripped into dilute sulphuric acid containing tartaric acid. Following the removal of bromine by evaporation of the solution, antimony(V) is reduced to antimony(III) with potassium iodide in approximately 3M hydrochloric acid and finally determined by hydride-generation atomic-absorption spectrometry at 217.8 nm with sodium borohydride as reluctant. Interference from platinum and palladium, which are partly co-extracted as xanthates under the proposed conditions, is eliminated by complexing them with thiosemicarbazide during the iodide reduction step. Interference from gold is avoided by using a 3M hydrochloric acid medium for the hydride-generation step. Under these conditions gold forms a stable iodide complex.

Determination of tellurium in ores, concentrates and related materials by graphite-furnace atomic-absorption spectrometry after separations by iron collection and xanthate extraction.

A method for determining approximately 0.01 mug/g or more of tellurium in ores, concentrates, rocks, soils and sediments is described. After sample decomposition and evaporation of the solution to incipient dryness, tellurium is separated from > 300 mug of copper by co-precipitation with hydrous ferric oxide from an ammoniacal medium and the precipitate is dissolved in 10M hydrochloric acid. Alternatively, for samples containing 300 mug of copper, the salts are dissolved in 10M hydrochloric acid. Tellurium in the resultant solutions is reduced to the quadrivalent state by heating and separated from iron, lead and various other elements by a single cyclohexane extraction of its xanthate complex from approximately 9.5M hydrochloric acid in the presence of thiosemicarbazide as a complexing agent for copper. After washing with 10M hydrochloric acid followed by water to remove residual iron, chloride and soluble salts, tellurium is stripped from the extract with 16M nitric acid and finally determined, in a 2% v/v nitric acid medium, by graphite-furnace atomic-absorption spectrometry at 214.3 nm in the presence of nickel as matrix modifier. Small amounts of gold and palladium, which are partly co-extracted as xanthates if the iron-collection step is omitted, do not interfere. Co-extraction of arsenic is avoided by volatilizing it as the bromide during the decomposition step. The method is directly applicable, without the co-precipitation step, to most rocks, soils and sediments.

An homologous radioimmunoassay for coho salmon (Oncorhynchus kisutch) vitellogenin, with general applicability to other Pacific salmonids.

This paper describes an homologous radioimmunoassay for coho salmon vitellogenin that demonstrates parallel cross-reactivity for plasma vitellogenin of all Pacific salmonids tested (chinook, chum, coho, pink, and sockeye salmon, and cutthroat and rainbow trout), but not for Atlantic salmon or two nonsalmonids: common carp and sablefish. Plasma vitellogenin levels were high in ovulatory female Pacific salmonids (micrograms/ml to mg/ml range), but were mostly nondetectable in spermiating males of the same species.

Estrogen-induced vitellogenin production by triploid coho salmon (Oncorhynchus kisutch), and its effect on plasma and pituitary gonadotropin.

Vitellogenin production was induced in immature diploid and triploid coho salmon by the weekly injection of 17 beta-estradiol at 1 mg/kg body wt. There was no significant difference between diploids and triploids for any of the results obtained, i.e., change in plasma vitellogenin and gonadotropin levels, hepatosomatic index, or pituitary gonadotropin content. Plasma vitellogenin levels were significantly higher in 17 beta-estradiol-treated fish than in sham-injected fish within a week of the first injection, and continued to rise with each subsequent injection. Plasma gonadotropin levels, on the other hand, were slightly (but significantly) depressed. The 17 beta-estradiol-treated fish had higher hepatosomatic indices and pituitary gonadotropin contents than sham-injected fish by 3 weeks after the first treatment. These data suggest that the occasional postmeiotic oocytes observed in triploids do not grow to full maturity due, in part, to an absent or diminished estrogen stimulus from the ovary on hepatic vitellogenin production.

Determination of cobalt, nickel, lead, bismuth and indium in ores, soils and related materials by atomic-absorption spectrometry after separation by xanthate extraction.

A method for determining approximately 0.5, mug/g or more of cobalt, nickel and lead and approximately 3 mug/g or more of bismuth and indium in ores, soils and related materials is described. After sample decomposition and dissolution of the salts in dilute hydrochloric-tartaric acid solution, iron(III) is reduced with ascorbic acid and the resultant iron(II) is complexed with ammonium fluoride. Cobalt, nickel, lead, bismuth and indium are subsequently separated from iron, aluminium, zinc and other matrix elements by a triple chloroform extraction of their xanthate complexes at pH 2.00 +/- 0.05. After the removal of chloroform by evaporation and the destruction of the xanthates with nitric and perchloric acids, the solution is evaporated to dryness and the individual elements are ultimately determined in a 20% v/v hydrochloric acid medium containing 1000 mug/ml potassium by atomic-absorption spectrometry with an air-acetylene flame. Co-extraction of arsenic and antimony is avoided by volatilizing them as the bromides during the decomposition step. Small amounts of co-extracted molybdenum, iron and copper do not interfere.

The growth and reproductive endocrinology of adult triploid Pacific salmonids.

This paper describes the effect of triploidy on growth and reproductive endocrinology in the months leading up to and including spawning in rainbow trout,Salmo gairdneri, and pink salmon,Oncorhynchus gorbuscha. Growth rates were the same for diploid and triploid rainbow trout, but triploid female pink salmon were smaller than maturing diploid females and diploid and triploid males of the same age. Triploid males of both species developed typical secondary sexual characteristics and had normal endocrine profiles, although their cycle appeared to be delayed by about one month. Triploid females remained silvery in appearance and showed no endocrine signs of maturation, even at the level of the pituitary. Thus, although triploids of both sexes are genetically sterile, only the females do not undergo physiological maturation.