Recombinant bovine growth hormone identification and the kinetic of elimination in rainbow trout treated by LC-MS/MS.
ABSTRACT The efficiency of the administration of recombinant bovine growth hormone (rbGH) to enhance fish growth has been widely reported in the literature. Although its use is probable and has been described in several countries, rbGH is prohibited in the European Union (Council Decisions 1994/936/EC , 1999/879/EC ). In this context, an analytical strategy was optimised in order to identify rbGH-treated fish. Currently, one of the most difficult challenges for the detection of rbGH in fish is probably the choice of the matrix and the corresponding available quantity for analysis. Therefore, based on a previous efficient protocol developed for mammalian species, a method was adapted for very limited serum volume (50 µl) and was successfully implemented to analyse serum collected from seven trout treated with rbGH. The detection of rbGH was possible from the very first day after administration and the hormone could easily be identified at least for 1 month with levels in the range 5-10 µg ml(-1). The limits of detection (LODs) estimated around 0.5 µg ml(-1) rbGH in fish serum are far below observed concentrations in incurred samples and therefore attest to the relevance of the developed protocol.
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ABSTRACT: Two hundred rainbow trout (Oncorhynchus mykiss, mean weight 301.5 g) were allotted to four treatments with five replicates in a randomized block design to determine the dose-response effects of recombinant bovine somatotropin (rbST; Posilac) on growth performance and carcass composition. Treatments were sham-injected controls (S), 10 micrograms/g BW of rbST (L), 20 micrograms/g BW of rbST (M), and 30 micrograms/g BW of rbST (H). The tanks held 135 L; water flow = 15.1 L/min; temperature = 15 degrees C. The fish were maintained in a 12-h light:dark cycle and hand-fed twice daily. The fish received a single intraperitoneal (i.p.) injection on d 0 and were weighed on d 0, 14, 28, and 56. On d 56 the fish were killed. The whole body (WBW), eviscerated carcass (EC), viscera (VIS), and reproductive organ weights and the increase in average daily body length (ADL) were determined. Recombinant bST reduced (linear, P < .004) feed intake 17.6% from d 0 to 14 and improved ADG 44.8% from d 0 to 14 (linear, P < .001) and 8.1% from d 0 to 56 (linear, P < .022). Treated groups had improved (linear, P < .001) feed efficiencies for d 0 to 28. Treatment with rbST increased final weight (linear, P < .018) and length (linear, P < .001), decreased carcass dry matter (linear, P < .001) and fat (linear, P < .001), content, increased carcass ash (linear, P < .001) and tended to increase carcass protein (linear, P < .054) content. Recombinant bST increased WBW (linear, P < .018) and EC (linear, P < .003) but decreased (linear, P < .015) testes weight. Ovary weights, VIS and overall gonadosomatic index were unaffected (P > .05) by rbST. Recombinant bST was undetectable in serum samples taken on d 56 as determined by radioimmunoassay. Overall, the improved ADG, feed efficiency, body mass, and composition indicate that administration of rbST to rainbow trout may be an efficacious method of modulating growth in fish.Journal of Animal Science 11/1995; 73(11):3216-22. · 1.92 Impact Factor
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ABSTRACT: Numerous studies demonstrated that rbST increased growth rates in several fish species, and several species exhibit GH production in tissues other than the pituitary. The role of tissue GH and IGF-I in regulating fish growth is poorly understood. Therefore an experiment was conducted to examine the effects of rbST treatment on tissue GH, IGF-I, and IGF-I receptor-A (rA) expression in rainbow trout. Rainbow trout (550 +/- 10 g) received either intra-peritoneal injections of rbST (120 microg/g body weight) or vehicle on days 0 and 21, and tissue samples were collected on days 0, 0.5, 1, 3, 7, and 28 (n = 6/day/trt). Total RNA was isolated and assayed for steady-state levels of IGF-I, IGF-IrA, and GH mRNA using quantitative RT-PCR. Insulin-like growth factor-I mRNA levels increased in liver, gill, gonad, muscle, brain, and intestine in response to rbST treatment (P < 0.10). Liver IGF-I mRNA increased (P < 0.01) 0.5 day after treatment and remained elevated throughout the trial. Intestine IGF-I mRNA increased (P < 0.05) in treated fish from day 1 to day 3, then decreased to day 7 and increased again at day 28, and remained elevated above control levels throughout the trial. Gill IGF-I mRNA levels increased (P < 0.05) 1 day after treatment and remained elevated throughout the trial. Heart IGF-IrA mRNA levels decreased (P < 0.05) while gonad GH mRNA levels increased (P < 0.10) following rbST treatment. These results demonstrate that rbST treatment increased IGF-I mRNA levels in extra-hepatic tissues, and decreased heart IGF-IrA and increased gonad GH mRNA levels. Because the primary source for endocrine IGF-I is liver, the increased IGF-I mRNA reported in extra-hepatic tissues may indicate local paracrine/autocrine actions for IGF-I for local physiological functions.General and Comparative Endocrinology 02/2004; 135(3):324-33. · 2.67 Impact Factor
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ABSTRACT: Equine growth hormone (eGH) has been available since 1998 as an approved drug (EquiGen-5, Bresagen) containing recombinant eGH (reGH). It is suspected of being illegally administered to racehorses in order to improve physical performance and to speed-up wound healing. Thus it may be considered a doping agent which would require a sensitive and reliable method of identification and confirmation in order to regulate its use in racehorses. reGH differs from the native eGH by an additional methionine at the N-terminal (met-eGH) and has never been unambiguously detected in any type of biological matrix at trace concentrations (1-10 microg/L). A plasma sample (4 mL) was treated with ammonium sulfate at the reGH isoelectric point and the pellet was purified by solid-phase extraction. Specific peptides were generated by trypsin digestion and analyzed by LC-MS/MS. The detection limit was 1 microg/L. The method was validated according to European Union regulation (DEC/2002/657/EC) and the Association of Official Racing Chemists (AORC) requirements. Furthermore, it was successfully applied to determining the plasma concentrations of reGH with time using linear ion trap mass analyzer. The presence of this prohibited hormone (reGH) was also successfully detected by triple quadrupole mass spectrometry up to 48 h postadministration of reGH to a horse. The present LC-MS/MS method is the first with adequate sensitivity and specificity for detection of reGH, rbGH, and endogenous eGH. Hence, an efficient analytical tool is proposed as a means to fulfilling the regulation of reGH abuse in the horse racing industry.Analytical Chemistry 10/2008; 80(21):8340-7. · 5.83 Impact Factor