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Black Bream Acanthopagrus Butcheri Is Improved by Treatment at Capture

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Black Bream Acanthopagrus Butcheri Is Improved by Treatment at Capture Aquaculture ELSEVIER Aquaculture 191 (2000) 351-366 — www.elsevier.nl/locate/aqua-online The efficacy of exogenous hormones in stimulating changes in plasma steroids and ovulation in wild black bream Acanthopagrus butcheri is improved by treatment at capture J.A. Haddy, N.W. Pankhurst* School o f Aquaculture and Co-operative Research Centre for Aquaculture, University o f Tasmania, PO Box 1214 Launceston, Tasmania 7250, Australia Received 23 June 1999; received in revised form 1 May 2000; accepted 26 May 2000 Abstract Sexually mature female black bream were captured by rod and line and injected with saline, human chorionic gonadotropin (hCG) or luteinizing hormone releasing hormone analogue (LHRHa) at capture, or 24 h post capture (saline and LHRHa treatments only). All fish were bled and checked for ovulation for 5 days post injection. Plasma levels of oestradiol (E2), testosterone (T), 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP) and cortisol were determined by radioimmunoas­ say. Saline-injected fish ovulated only on day 1, whereas treatment with LHRHa or hCG resulted in fish ovulating throughout the experiment. Treatment with LHRHa at capture resulted in a better ovulatory response than treatment with hCG at capture or LHRHa 24 h post capture. Plasma E2 levels in saline-injected fish were high at capture and had significantly dropped 1 day after capture. Injection with hCG or LHRHa at capture resulted in plasma E2 levels remaining significantly elevated for 2 days post injection. Injection of LHRHa 24 h post capture failed to significantly elevate plasma E2 levels over controls. Plasma T levels were similar to E2 profiles. Plasma levels of 17,20ßP were not significantly different between any treatments, but showed a Corresponding author. Tel.: +61 363 24 3801 ; fax: +61 363 24 3804. E-mail address: [email protected] (N.W. Pankhurst). 0044-8486/00/$ - see front matter ©2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 -8 4 8 6 (0 0 )0 0 4 4 5 -2 352 J.A. Haddy, N.W. Pankhurst/ Aquaculture 191 (2000) 351-366 tendency to increase after capture. Plasma cortisol levels showed no treatment effects and were initially low at capture before becoming elevated between days 1 and 2 post capture. These results show that capture and handling stress reduce the responsiveness of fish to exogenous hormone treatment and that best results are obtained if hormonal treatment is administered at the time of capture. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Induced ovulation; Reproduction;Acanthopagrus butcheri', Stress; Cortisol; Oestradiol; Testos­ terone 1. Introduction One of the requirements in developing new species for aquaculture is obtaining reliable quantities of viable eggs. In the initial development stages of a new species with aquaculture potential, eggs are usually obtained by the capture and hormonal induction of ovulation of wild-caught females. This approach commonly involves the capture and transportation of fish to holding facilities, with fish being treated with exogenous hormones some time after capture. Although the effects of exogenous hormones on ovulatory and endocrine events have been examined in detail in many species (see reviews by Lam, 1982; Donaldson and Devlin, 1996; Peter and Yu, 1997), few studies have investigated the effects of a delayed treatment on subsequent endocrine and ovulatory responses. This is likely to be a particular problem in species that are particularly sensitive to stress, where plasma levels of oestradiol (E2) and testosterone (T) are rapidly depressed within 1 h of capture (Carragher and Pankhurst, 1991; Jardine et al., 1996; Cleaiy, 1998; Haddy and Pankhurst, 1999). However, it is currently unknown whether stress-induced disruption of the hypothalamic-pituitary-gonad axis affects the subsequent ability of fish to respond to exogenous hormone treatment. Induced ovulation protocols in marine fish most commonly utilise human chorionic gonadotropin (hCG) or luteinizing hormone releasing hormone analogues (LHRHa). LHRHa stimulates the release of endogenous gonadotropin (GtH), whereas, hCG mimicks endogenous GtH (reviewed in Donaldson and Devlin, 1996). In some species, hCG has a low biopotency and high doses or multiple injections are required, and this has led to a more widespread use of LHRHa (Donaldson and Devlin, 1996). The Australian sparid Acanthopagrus butcheri (black bream) is currently under investigation as an aquaculture candidate for inland saline water culture. Initial stages of culture are likely to be dependent on hormonal manipulation of sexually mature fish captured from the wild. Given that this species shows acute and profound sensitivity to stress (Haddy and Pankhurst, 1999), this study investigated whether delay after capture changes the efficacy of treatment with LHRHa at inducing changes in plasma levels of gonadal steroids, and the occurrence or frequency of ovulation. The ovulatory response of fish to treatment and handling was assessed in terms of the number of ovulations, and the quantity and quality of the eggs produced. In the absence of a specific GtH assay for black bream, the endocrine response of fish to treatment and handling was assessed by measuring plasma levels of cortisol, E2, T and 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP). The possibility that fish were differentially responsive to hCG or LHRHa J.A. Haddy, N.W. Pankhurst/ Aquaculture 191 (2000) 351-366 353 Table 1 Summary of the percentage of fish ovulating for the duration of experiments 1 and 2 Treatments Time (days) 0 1 2 3 4 5 Experiment 1 At capture Saline 0 57 0 0 0 0 LHRHa 0 28 57 66 25 33 24-h post capture Saline 0 43 0 0 0 0 LHRHa 0 22 11 0 33 16 Experiment 2 Saline 0 57 0 0 0 0 LHRHa 0 86 86 100 100 0 HCG 0 62 37 75 71 33 was assessed in a second experiment once the appropriate treatment window was determined. 2. Materials and methods Sexually mature female black bream were ipturedca by rod and line from the Meredith (148°7'S, 42°4'E) and Swan River estuaries (148°4'S, 42°4'E) at Swansea, Tasmania. Fish were blood-sampled by caudal puncture using heparinized syringes, fin clipped for individual identification and the time of hooking and blood-sampling recorded. Blood was stored on ice, plasma obtained by centrifugation, then frozen and stored at — 18°C Table 2 Summary of the proportions of fish ovulating, and quality of eggs collected experimentsin 1 and 2 Treatments Proportion Proportion of Mean number Mean of ovulating fish serially of ovulations fertilisation fish (%) ovulating (%) per fish3 (%)b Experiment 1 At capture Saline 57 0 1 0(1) THRHa 85 57 2.4 60 + 16.5 (3) 24-h post capture Saline 43 0 1 98(1) THRHa 44 22 1.5 20(1) Experiment 2 Saline 57 0 1 43.0 + 22.4 (3) THRHa 100 100 3.14 46.9 + 7.4 (16) HCG 87.5 75 2.86 41.1+7.7 (9) a Calculated from ovulating fish only. bFish numbers in parentheses. 354 J.A. Haddy, N.W. Pankhurst/ Aquaculture 191 (2000) 351-366 until required for assay. Fish were placed in 400-1 plastic tanks with oxygenation until transportation to the laboratory (3-20 h experiment 1; 3-7 h experiment 2), where they were placed in 1000-1, temperature-controlled (18-20°C) tanks supplied with recirculat­ ing seawater. 2.1. Experiment 1: effect of delayed injection time Fish were caught from October to November 1997 between 17:35 and 20:50 h or 5:45 and 13:34 h. Sixteen fish (mean weight = 564 ± 50 g) were blood-sampled at capture without anaesthesia, placed into the holding tanks and transported to the laboratory. Twenty four hours after capture, fish were anaesthetised in 0.05% 2-pheno- (a) 500 -i (2) ] saline (4) (3) LHRHa 400 - (6) (6 ) (3) 300 (7) be 1 (7) 200 - (7) be V U i be (7) ■ 100 - ab I ■ ,7) (7)a I o I </> (b) r 500 -i o o 400 - re (6) E (7) (4) re 300 re (6 ) CL 200 - (8) (6 ) (7) (9) b l (9) 100 - (7) (9) Í a a i 0 m . DAYS Fig. 1. Plasma cortisol concentrations (mean + SE) in black bream injected (indicated by arrow) with either saline or 50 |jLg kg-1 LHRHa, at capture (a), or 24-h post capture (b). Values that are significantly different (P < 0.05) have different letters; values indicated by (-) were not included in the analysis due to lown values (given in parenthesis). J.A. Haddy, N.W. Pankhurst/ Aquaculture 191 (2000) 351-366 355 xyethanol, blood-sampled and biopsied for macroscopic gonad condition (Haddy and Pankhurst, 1998). Fish that contained mature vitellogenic oocytes in the biopsy were then weighed, injected intraperitoneally with 50 pig kg-1 body weight of 50 pig ml-1 des-Gly10 (D-Ala6)-luteinizing hormone releasing hormone ethylamide (LHRHa) (Sigma) (n= 9) or 1 ml kg-1 of teleost saline (n=7) and checked for ovulation. Treatments were allocated on an alternating basis. LHRHa- and saline-treated fish were combined into tanks with 3-4 male fish. Ovulated females were manually stripped and the eggs fertilised in seawater of 35%o salinity, using fresh sperm pooled from 3-4 males. Males were stripped by wiping dry the genital papilla region and milt expressed using slight abdominal pressure. The first portion of stripped milt was not collected to ensure milt was not contaminated with urine. Milt was collected while being expressed into dry 5-ml syringes and placed on ice until use. Fertilised eggs were viewed under a dissecting microscope and the fertility (division to 2-8-cell stage) of the first 100 eggs encountered recorded.
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