The use of stable isotopes to investigate the effects of supplemental lysine and methionine on protein turnover and amino acid utilization in pacu, Piaractus mesopotamicus, juveniles Eduardo Gianini Abimorad a , Carlos Ducatti b , Daniela Castellani a , Rosângela Kiyoko Jomori c , Maria Célia Portella d , Dalton José Carneiro d, ⁎ a APTA/SAA-SP — Agência Paulista de Tecnologia dos Agronegócios, Pólo Regional Noroeste Paulista, Cx. Postal 61, 15500-970 Votuporanga, SP, Brazil b UNESP — Univ. Estadual Paulista, Centro de Isótopos Estáveis, Distrito de Rubião Junior s/n, 18618-970 Botucatu, SP, Brazil c FAFRAM/FE - Faculdade Dr. Francisco Maeda/Fundação Educacional, 14500-000 Ituverava, SP, Brazil d UNESP — Univ. Estadual Paulista, Centro de Aquicultura, Via Prof. Paulo Donato Castellane s/n, 14884-900 Jaboticabal, SP, Brazil abstract article info Article history: Received 12 March 2013 Received in revised form 3 June 2014 Accepted 7 June 2014 Available online 14 June 2014 Keywords: Plant protein Supplementation Deficiency Amino acid Protein degradation Muscle The present study investigated the use of the stable isotope technique to evaluate the effects of nutritionally de- ficient diets, supplied or not with lysine and/or methionine, on the isotopic turnover of bio-elements (carbon and nitrogen) in the muscle tissue of pacu juveniles. In addition, it evaluated the contributions of growth rate (k) and metabolic rate (m) to isotopic turnover. Fish with initial weight of 10 g were fed 4 experimental diets for 100 days: a nutritionally deficient diet without amino acid supplementation (AAd) or supplemented with lysine (Lys), methionine (Met) and lysine–methionine (LysMet). During this period, muscle samples were taken from the fish to analyze isotopic signature and calculate δ 13 C and δ 15 N turnover rates. Fish fed Lys and LysMet diets ex- hibited higher weight gain, growth rate, feed consumption and apparent feed conversion (P b 0.05) than fish fed AAd. Fish supplemented with Met displayed intermediate growth performance. The exponential model applied indicated that fish fed Lys and LysMet diets had the lowest turnover rates and discrimination factors (Δ), partic- ularly for δ 15 N. Lower half-life for δ 15 N was observed in fish fed AAd and Met diets. As indicated by the stable iso- tope technique, dietary lysine supply reduces muscle protein catabolism (m) and improves growth (k) in pacu juveniles fed plant protein-based diets. Therefore, we proved that the stable isotope technique, particularly 15 N, is a useful tool in nutritional studies, providing information on isotopic turnover rates. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Protein turnover rates in fish have been studied in rainbow trout (Peragón et al., 1994, 2001; Smith et al., 2001), sea bass Dicentrarchus labrax (Langar and Guillaume, 1994; Langar et al., 1993), carp (de la Higuera et al., 1998) and eel Anguilla anguilla (de la Higuera et al., 1999) as a function of factors such as water temperature, food depriva- tion, refeeding, feeding frequency and especially diet composition. The abovementioned studies accessed the fractional protein- turnover rates in the muscle of the test animals using the radioisotope ( 3 H-tritium)-marked phenylalanine injection method. However, this technique is limited to short-term tracing of dietary components. Another procedure, employing natural stable isotopes, is a safer alterna- tive for evaluating the long-term contribution of dietary components in animal tissues (Schroeder, 1983), allowing us to determine the turnover rates of these elements. This can be obtained due to the stable relation- ship between natural isotopes (such as 13 C/ 12 C and 15 N/ 14 N), which show characteristic variations in plant tissues and algae. The values of δ 13 C in animal tissues are similar to those in animal diet, with only ± 1‰ difference caused by the small isotope partitioning associated to respiration and carbon incorporation by the animal (Créach et al., 1997; DeNiro and Epstein, 1978). On the other hand, the δ 15 N differences between animal tissues and diets are greater, reaching ±3–4‰. Nitrogen fractionation is high because it un- dergoes deamination and amino acid decarboxylation, and animals fed low-quality diets display higher isotope partitioning or 15 N en- richment (Gamboa-Delgado and Le Vay, 2009; Roth and Hobson, 2000; Waddington and MacArthur, 2008). Plants exhibit different nitrogen isotopic signatures according to their specific composition (Gannes et al., 1998). Additionally, δ 15 N values of animal food sources differ from those of plant food sources, Aquaculture 433 (2014) 119–124 ⁎ Corresponding author at: UNESP/CAUNESP — Universidade Estadual Paulista, Centro de Aquicultura, Via Prof. Paulo Donato Castellane s/n, 14884-900, Jaboticabal, SP, Brazil. Tel.: +55 16 32092615; fax: +55 16 32032268. E-mail address: daltonjc@caunesp.unesp.br (D.J. Carneiro). http://dx.doi.org/10.1016/j.aquaculture.2014.06.006 0044-8486/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online