Steroidogenic capacity of coho salmon ovarian follicles throughout the periovulatory period

Coho salmon follicles obtained at various times throughout the periovulatory period were incubatedin vitro with graded amounts of partially purified salmon gonadotropin (SG-G100) for 24 h and the amounts of 17β-estradiol, testosterone and 17α20β dihydroxy-4-pregnen-3-one (17α20βP) released into the media determined by radioimmunoassay. By this approach, the pattern of steroid secretion by ovarian follicles was shown to change in relation to the developmental status of the oocyte. Full-grown immature follicles produced large amounts of 17β-estradiol but negligible amounts of testosterone and 17α20βP. Both basal and gonadotropin-stimulated 17β-estradiol production was subsequently reduced with advancing oocyte development. In contrast, the production of testosterone and 17α20βP increased during the course of ovarian development with testosterone production highest in follicles with a peripheral germinal vesicle and 17α20βP production highest in matured and postovulatory follicles. These data are discussed in relation to information on the preovulatory changes in circulating levels of steriod hormones in salmonids.     

Steroid release from separated theca and granulosa layers of Atlantic salmon (Salmo salar) ovarian follicles: the effects of a purified salmon gonadotrophin preparation

Theca and granulosa layers were removed from ovarian follicles of mature Atlantic salmon (Salmo salar) and were separately incubated under sterile conditions with and without a partially purified salmon gonadotrophin preparation (GTH). Aliquots of the incubation media were removed at intervals and analysed for the steroids 17, 20-dihydroxy-4-pregnen-3-one (1720P), 17-hydroxyprogesterone, progesterone, androstenedione, testosterone and oestradiol. The biosynthesis of C19 and C21 steroids was very largely restricted to the thecal tissue and was markedly stimulated in the presence of GTH. Androstenedione (max 65 ng/ml) and testosterone (max 14 ng/ml) were released from the earliest stages of incubation whereas the release of 17-hydroxyprogesterone (max 51 ng/ml) and progesterone (max 5.5 ng/ml) commenced only after a lengthy induction period. A trace (1.0 ng/ml) of 1720P was produced by the theca in the presence of GTH but oestradiol was not detected. The granulosa preparations released levels of 17-hydroxyprogesterone and androstenedione only marginally above the detection limits (ca 0.7 ng/ml) and there was little stimulation of output with GTH. Oestradiol (max 4 ng/ml) was released only in the presence of GTH. 1720P, progesterone and testosterone were not detected as products of this tissue. These results, together with those derived earlier from incubations of complete follicles support the view that the synthesis of 1720P is essentially a two-cell process in which 17-hydroxyprogesterone produced in the theca is subject to the action of steroid 20-hydroxysteroid dehydrogenase in the granulosa. The temporal pattern of release of steroids in these and earlier experiments is considered in relation to mechanisms of steroid biosynthesis and to their possible roles in oocyte final maturation.     

Effects of exotic salmonids on juvenile Atlantic salmon behaviour

Abstract –  We examined the effects of two salmonid species, chinook salmon ( Oncorhynchus tschwaytscha ) and brown trout ( Salmo trutta ), both exotic species to Lake Ontario, on behaviour and foraging success of juvenile Atlantic salmon ( S. salar ), a native species to Lake Ontario, in an artificial stream. We found that both exotic species have effects on Atlantic salmon behaviour, but that neither had an effect on foraging success. These results may explain why the Atlantic salmon re-introduction programme in Lake Ontario has had little success, as more than 3 million exotic salmonids are released in Lake Ontario streams annually.     

In vitro steroidogenesis by testicular fragments and ovarian follicles in a hybrid sturgeon, Bester

In this study, developmental changes in the steroidogenic capacity of testicular fragments and isolated ovarian follicles of a hybrid sturgeon, Bester, at a variety stage of developments were examined. Testicular fragments or isolated ovarian follicles were incubated in L-15 medium in the presence or absence of different concentrations of five preparations; forskolin, human chorionic gonadotropin (HCG), pregnenolone (P₅), 17-hydroxyprogesterone (17OHP) and testosterone (T) for 18 h at 15 °C. After incubation, concentrations of 11-ketotestosterone (11 KT) (testis) and, 17-estradiol (E₂) (ovarian follicles) and 17,20-dihydroxy-4-pregnen-3-one (DHP) (testis and ovarian follicles) were measured. 11KT was detected in the media following incubation with P₅, 17OHP and T. Its concentration was higher during late spermatogenesis and prespermiation and lower at the degeneration stage. Both P₅ and 17OHP were converted to DHP during the prespermiation stage. Forskolin had little stimulatory effect on the synthesis of 11KT and DHP and HCG did not induce the production of these steroids.E₂ was detected in the medium following incubation of follicles with P₅, 17OHP and T at all stages of oocyte development. The concentration of E₂ in the medium increased during vitellogenesis with the peak production occurring at the tertiary yolk stage. In contrast, the potencies of follicles to produce steroids shifted to the production of DHP during migratory nucleus stage. Forskolin and HCG had little effect on the synthesis of E₂ and DHP. These results demonstrated that the failure of spontaneous spermiation or ovulation is not due to the insufficient synthesis of DHP, but may due to the lack of availability of precursors.     

Melatonin, but not somatostatin-14 influences in vitro steroidogenesis by ovarian follicles of rainbow trout

Ovarian follicles taken from sexually maturing rainbow trout at the mid-vitellogenic stage of ovarian development were incubated in vitro in the presence or absence of melatonin or somatostatin-14 (SRIF-14) to determine whether there is evidence of a direct action of these factors on gonadal steroidogenesis in fishes. The steroidogenic capacity of the ovarian follicles was assessed by measuring testosterone (T) and 17-estradiol (E₂) release into the incubation medium, and by examining the steroid metabolites produced following incubation of follicles with radiolabelled steroid precursors.Melatonin appears to elicit a biphasic effect on steroidogenesis by in vitro rainbow trout ovarian follicles; at a concentration of 1 × 10^–3 M, melatonin stimulated basal T and E₂ production, but at a concentration of 1 × 10^–2 M there was an inhibition of basal and sGtH-stimulated T and E₂ Melatonin may act to reduce the activity of specific steroidogenic enzymes, since there was evidence of melatonin at 1 × 10^–2 M enhancing the accumulation of [³H]17-hydroxyprogesterone in the medium following incubation with [³H]pregnenolone, possibly suggesting the inhibition of C_17,20-lyase activity. In contrast, SRIF-14, used at concentrations of 1 × 10^–8 M and 1 × 10^–6 M, had no effect on basal or sGtH-stimulated E₂ or T production by ovarian follicles, incubated in vitro.     

Insulin binding to isolated hepatocytes of Atlantic salmon and rainbow trout

The questions addressed in this study were: 1) whether insulin added to the incubation medium can down-regulate ¹²⁵I insulin binding to isolated hepatocytes of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss); 2) whether quantitative assessment of insulin processing can be made on isolated fish liver cells; 3) how ambient temperatures can affect insulin binding, and down-regulation of insulin receptors. After isolation and a short (up to 4h) “metabolic recovery period”, liver cells were used either directly in ¹²⁵I insulin binding assay or first preincubated for 18h at 4°C or for 3h at 15°C, with or without mammalian or salmon insulin in concentrations ranging from 1 to 1000 nM. Preincubation at 15°C, decreased binding capacity (number of binding sites per liver cell) in all five independent hepatocyte preparations treated with 1000 nM insulin and in four out of five preparations treated with 100 nM insulin. At 4°C insulin binding sites were down-regulated in less than 50% of all hepatocyte preparations and only in the presence of 1000 nM insulin. Differential quantitive assessment was made of a) intact free insulin; b) insulin degraded; c) intact insulin bound to the cell membrane; d) internalized but degraded insulin, and e) intact insulin internalized by liver cells. Hepatocytes preincubated with 100 – 1000 nM insulin at 15°C bound and internalized less ¹²⁵I insulin. We hypothesize that in vivo, at water temperatures of 15°C and higher, extreme physiological levels of plasma insulin may regulate the numbers of insulin receptors in the salmonid liver. In contrast, in fish inhabiting cold waters the regulation of insulin receptors by circulating plasma insulin seems to be of little physiological importance. Presented in part at the Western Regional Conference on Comparative Endrocrinology, Tempe, Arizona, U.S.A., 1991 and at the Meeting of Italian Society of Experimental Biology, Sorrento, Italy, 1991. Supported by grants from NSF of the USA#DCB 8915935 to E.M.P., NSERC of Canada OPGA 6944 to T.W.M., North Atlantic Treaty Organization (NATO) grant #0926/87 to C.O. and E.M.P., and CYCIT grant of Spain to J.G.     

Further evidence of the equivocal effects of cortisol on in vitro steroidogenesis by ovarian follicles of rainbow trout Oncorhynchus mykiss

Previous studies on salmonids have yielded equivocal results as to the role of cortisol in directly inhibiting ovarian steroidogenesis. In an effort to determine why this might be so, isolated ovarian follicles of rainbow trout were incubated with and without cortisol under varying conditions of gonadotropin or steroid precursor stimulation, incubation time and temperature. Cortisol at concentrations of 100–1000 ng ml–1 suppressed basal production of 17-estradiol (E2) in only 4 out of 20 experiments, had no effect on human chorionic gonadotropin (hCG)-stimulated production, and no suppressive effect on 17-hydroxyprogesterone (17P)-stimulated production in 14 experiments, but increased E2 production in response to 17P in 2 experiments. Cortisone had no effect on basal E2 production, suggesting that cortisol was unlikely to be exerting any effect indirectly after further metabolism. Extended incubation times at 12 °C resulted in overall decreased levels of E2 in incubation media, but this had no obvious effect on patterns generated by treatment with cortisol. Extended incubation at 18 °C did change the pattern of response to treatment with cortisol in 1 out of 3 experiments. All incubations examined produced substantial amounts of E2-glucuronide but this showed no obvious relationship to whether or not inhibition of E2 production by cortisol was observed. Effect of stress history was examined by incubating follicles from stressed or unstressed fish. In follicles from fish nearing the end of vitellogenesis, stress resulted in reduced production of both testosterone and E2 in response to hCG, but increased conversion of 17P to E2. The same effect was not observed in follicles from fish at an earlier stage of vitellogenesis. Measurement of E2 uptake by follicles from selected experiments showed that follicles contained considerable amounts of E2 and were potentially a sink for steroid produced during incubation. The experiments show that a consistent effect of cortisol on ovarian steroidogenesis remains elusive, but that stimulatory effects are as likely to occur as inhibitory effects. All responses are potentially further confounded by loss of free steroid from the medium by conjugation or absorption into the oocytes.     

Chinook salmon impede Atlantic salmon conservation in Lake Ontario

Abstract – Non-native species can have substantial impacts on successful restoration of native species. Here, we examined effects of chinook salmon ( Oncorhychus tshawytscha ), an exotic species introduced to Lake Ontario to enhance recreational angling, on reintroduced Atlantic salmon ( Salmo salar ) in a Lake Ontario tributary stream. Field enclosure studies revealed that adult Atlantic salmon activity rate was elevated, nest establishment delayed and mortality rates higher in the presence of chinook salmon. These results suggest that chinook salmon in Lake Ontario streams during fall spawning could impede successful re-establishment of Atlantic salmon in the lake.     

Energy use in spawning Atlantic salmon

Abstract – We studied some of the factors that might influence energy use in spawning Atlantic salmon (Salmo salar). Single females were placed into an experimental channel with either one or three males, after which spawning was monitored continuously. Male status was confirmed using genetic parentage analysis. Daily fat loss was monitored with the Torry Fish Fatmeter and validated through biochemical analyses. Several comparisons were in the expected direction but not statistically significant and therefore require further study: daily fat loss appeared higher for dominant males relative to subordinate males and in the three‐male treatment relative to the one‐male treatment. Most of the variation among individuals remained unexplained, suggesting that several as yet unknown factors strongly influence fat loss in spawning salmon. A large and significant effect was that daily fat loss was higher for females than for males, a difference that might contribute to the shorter spawning duration typical of females.     

Atlantic salmon in the North Pacific

The first catches of Atlantic salmon, Salmo salar L., in British Columbia (BC) waters occurred in 1987. The first reported escape of Atlantic salmon (2000 individuals) occurred in 1988. From 1988 to 1995, 97 799 Atlantic salmon were reported escaped from net pens in BC but the true number was higher as not all escapes are reported. Since 1987 a total of 9096 Atlantic salmon was caught in the coastal marine waters of BC, Washington and Alaska, and 188 were caught in fresh water. Most catches occurred in the Johnstone Strait area, where the abundance of salmon farms is highest. The most distant recovery occurred in 1994 when an Atlantic salmon was caught near the western end of the Alaska Peninsula. There have been no reports of successful reproduction of Atlantic salmon in the wild and no feral juveniles have been found. Atlantic salmon caught in the ocean in BC have substantial amounts of adipose tissue and they are heavier at length than fish caught in Alaska. The proportion of fish with prey items in their stomachs is generally low but higher in Alaska (13.1%) than in BC (5.8%). Most fish caught in fresh water are either maturing or mature.     

Effects of various dietary factors on astaxanthin absorption in Atlantic salmon (Salmo salar)

The experiment was designed to investigate the dietary factors that might enhance or interfere with astaxanthin (Ax) absorption in salmon including potentially interfering factors such as certain carotenoids (zeaxanthin and lutein), plant sterols, fibre and enhancing compounds such as cholesterol and vitamin E. Two hundred and eighty‐eight salmon (778 ± 78 g) were reared in sea water under controlled conditions and fed practical experimental diets. The experimental diets were supplemented with 40 mg Ax kg^?1, in addition to various dietary factors, including cholesterol (2%), vitamin E (450 IU kg^?1), wheat bran (5%), lutein (40 mg kg^?1), zeaxanthin (40 mg kg^?1) and phytosterol (2%). After 26 days of feeding, blood was collected and plasma was separated to determine the plasma Ax concentration. Ax was not detected in the plasma of fish fed the non‐pigmented diet. Fish fed diet containing 2% cholesterol significantly improved Ax absorption, which was reflected in the higher Ax concentration in plasma of Atlantic salmon. Other supplements including vitamin E, wheat bran, lutein, zeaxanthin and phytosterols in diet had no significant effect on plasma Ax concentration . Fish fed diet containing 2% cholesterol significantly increased cholesterol concentration in fish plasma. Phytosterol had no benefit to lower cholesterol plasma level in fish fed 2% phytosterol‐supplemented diet.     

Effects of hook and release on Atlantic salmon in the River Alta, northern Norway

The purpose of the study was to collect information on angling procedures and the effects of hook and release on Atlantic salmon in the River Alta, northern Norway, covering both grilse and multi-sea-winter salmon in a non-artificial setting with real anglers. Information on the angling procedure, handling of the fish and the condition of the fish at release was collected for individual salmon in catch logs (n=543, mean body length 82 cm), whereas physiological stress was studied in a sub-sample (n=15, mean body length 77 cm). To study post-release behaviour, survival and recapture rates, salmon were tagged with radio transmitters (n=30, mean body length 83 cm) and anchor T-tags (n=353, mean body length 79 cm). To evaluate the effects of the hook and release programme on the salmon population, number of spawning redds were recorded from a helicopter in 6 years during 1989–2000.

The results showed that at water temperatures 10.0–14.5 °C, a high proportion of the radio tagged salmon (97%) survived hook and release and stayed in known spawning areas during the spawning period. However, the behaviour after release seemed to be affected by hook and release. Only a small proportion (4%) of the anchor T-tagged salmon was caught more than once within the same season. Increased playing time, increased number of runs during the angling event, hooking in the throat, bleeding at the hook wound, increased handling time, air exposure and water temperature were factors that affected hooked and released Atlantic salmon negatively, either indicated by a poor condition at release, increased stress levels or unnatural behaviour after release. Number of spawning redds were more than doubled after the introduction of compulsory release of all angled salmon in Sautso (the upper 16% of the watershed inhabited by salmon) in 1998, which indicates that hook and release can be an effective management tool to enhance declining Atlantic salmon populations.     

Effects of decreases in photoperiod on growth and bimodality in Atlantic salmon Salmo salar L.

Two experiments were performed to study the relative significance of the absolute daylength and the change in photoperiod on the growth and development of bimodality in juvenile Atlantic salmon Salmo salar L. In Experiment A juveniles were reared on 24 h daily light until they were seven months old (65–82 mm in length after size grading). They were then divided into six groups and subjected to six photoperiods (6, 9, 12, 15, 18 and 24 h of light). In Experiment B the decrease in photoperiod was made in two steps. First, the day length was reduced to 18 and 21 hours, three months after first feeding when the weight of the juveniles averaged 2.5 g and one group was kept under 24 hour daily light. Two months later, each of these treatment groups was subdivided to produce new groups of juveniles (65–82 mm in length) under 6, 9 and 12 h of daily light. Irrespective of whether the photoperiods were reduced in one or two steps, groups held under short-day photoperiods, 6–12 h, grew significantly slower (Exp. A) and showed higher proportions of lower modal group fish (Exp. B) than groups treated with long-day photoperiods, greater than 12 h. There were low proportions of lower modal group fish among juveniles larger than 75 mm at the dates of decreases in daylength irrespective of photoperiod (Exp. B, 0–16%), and high or variable proportions among fish smaller than 75 mm, depending on photoperiod (Exp B. 32–71%). It is concluded that the growth response of juvenile Atlantic salmon changes in the range of 12–15 hours of daily light. This mechanism is probably linked to the size of the parr and is one important reason for the development of bimodal length-frequency distributions.     

Antibiotic resistance of Aeromonas salmonicida isolated from Atlantic salmon, Salmo salar L., in Scotland

Abstract. Antibiotic sensitivity patterns of 304 isolates of Aeromonas salmonicida from 229 outbreaks of furunculosis among salmon in Scotland between 1988 and 1990 were investigated. Fifty-five per cent were resistant to oxytetracycline and 37% resistant to oxolinic acid. Multiple resistance was common (52%) and 18 out of 19 antibiograms which were found in the first year recurred in the succeeding year. More than a quarter of the outbreaks were associated with two or more A. salmonicida variants distinguishable by their antibiotic sensitivity patterns. The implications of these findings in the control of furunculosis are considered.     

Surface properties of Streptococcus phocae strains isolated from diseased Atlantic salmon, Salmo salar L

Streptococcus phocae is an emerging pathogen for Chilean Atlantic salmon, Salmo salar, but the factors determining its virulence are not yet elucidated. In this work, cell surface–related properties such as hydrophobicity and haemagglutination, adhesion to mucus and cell lines, capsule detection, survival and biofilm formation in skin mucus and serum resistance of the isolates responsible for outbreaks in Atlantic salmon and seals were examined. Adhesion to hydrocarbons and the results of salt aggregation tests indicated most of the S. phocae were strongly hydrophobic. All isolates exhibited a similar ability to attach to the Chinook salmon embryo (CHSE) cells line, but were not able to enter CHSE cells. Haemagglutination was not detected. Our data clearly indicate that S. phocae can resist the killing activity of mucus and serum and proliferate in them, which could be associated with the presence of a capsular layer around the cells. Pathogenicity studies using seal and fish isolates demonstrated mortality or pathological signs in fish injected only with the Atlantic salmon isolate. No mortalities or histopathological alterations were observed in fish injected with extracellular products.     

Effects of aromatase inhibitors on sexual maturation in Atlantic salmon,Salmo salar,male parr

Atlantic salmon, Salmo salar, male parr were implanted with Silastic capsules filled with different aromatase inhibitors: 1,4,6-androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4OH), and the non-steroidal CGS16949 A, 4-benzonitrile monohydrochloride (CGS). Aromatization in brain homogenates were lower in salmon implanted with CGS and ATD than in controls. This was not the case for 4OH, but administration of 4OH to brain homogenates reduced the aromatase activity. All three aromatase inhibitors had effected gonadal weights in fish sampled in the summer, but the effects were markedly different among inhibitors. Plasma levels of the androgen 11-ketotestosterone (11KT) and the progestin 17, 20-dihydroxy-4-pregnen-3-one (17,20P) were measured by means of radioimmunoassay. CGS and ATD, but not 4OH, significantly decreased the plasma 17,20P levels in the autumn. Plasma levels of 11 KT were not influenced by ATD or CGS treatment, but 4OH had a lowering effect in one autumn sampling. ATD and 4OH (CGS not tested) increased the proportion of maturing males.These findings suggest that aromatization is of physiological importance in different mechanisms controlling reproduction in salmon.     

Effects of thyroid hormone on gonadotropin-induced steroid production in medaka,Oryzias latipes,ovarian follicles

Blood and ovarian samples were collected at intervals of 4h prior to spawning time from medaka (Oryzias latipes) that were maturationally synchronized with artificial photoperiod (14h light: 10h dark). Plasma estradiol-17β (E₂) levels increased rapidly from 16h before spawning and peaked at 8h before spawning. Follicle-enclosed oocytes (ovarian follicles) at different stages of development were isolated from the ovaries and used to study the in vitro effects of thyroid hormone (triiodothyronine; T₃) on pregnant mare serum gonadotropin (GTH)-induced E₂ production. GTH at a concentration of 100 IU/ml stimulated E₂ production by ovarian follicles collected between 32 and 16h before spawning. At 32h before spawning, T₃ (5 ng/ml) administered along with GTH (100 IU/ml) resulted in a 3.5 fold increase in E₂ production, compared with GTH administered alone. These results suggest that T₃ can act on ovarian follicles directly to modulate GTH-stimulated E₂ production in the medaka.     

Plasmid-mediated antibiotic resistance in Aeromonas salmonicida isolated from Atlantic salmon, Salmo salar L., in Scotland

Abstract. Forty oxytetracycline-resistant isolates of Aeromonas salmonicida obtained from outbreaks of furunculosis in Atlantic salmon in Scotland were tested for their susceptibility to 12 antibacterial agents. There were 10 resistance patterns with multiple resistance to two to six antibacterial agents. Transferable R-plasmids encoding oxytetracycline resistance were demonstrated in 11 out of the 40 isolates. The resistance transferred was multiple; to oxytetracycline, streptomycin, sulphamethoxine and trimethoprim, or to oxytetracycline and one or two of these in combination. Oxytetracycline resistance was transferred in a single, large step, >250-fold increase, and the MICs for individual transconjugants from mating populations were the same.     

Genetic characterization of Streptococcus phocae strains isolated from Atlantic salmon, Salmo salar L., in Chile

Streptococcus phocae is a beta-haemolytic bacterium frequently involved in disease outbreaks in seals causing pneumonia or respiratory infection. Since 1999, this pathogen has been isolated from diseased Atlantic salmon, Salmo salar , causing serious economic losses in the salmon industry in Chile. In this study, we used different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), repetitive extragenic palindromic PCR (REP-PCR) and restriction of 16S–23S rDNA intergenic spacer regions to evaluate the genetic diversity in S. phocae . Thirty-four strains isolated in different years were analysed. The S. phocae type strain ATCC 51973^T was included for comparative purposes. The results demonstrated genetic homogeneity within the S. phocae strains isolated in Chile over several years, suggesting the existence of clonal relationships among S. phocae isolated from Atlantic salmon. The type strain ATCC 51973^T presented a different genetic pattern with the PFGE, RAPD, ERIC-PCR and REP-PCR methods. However, the fingerprint patterns of two seal isolates were distinct from those of the type strain.