Aquaculture 338–341 (2012) 228–236

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Aquaculture

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Effects of feeding level and sexual maturation on carcass and fillet characteristics and indices of protein degradation in rainbow trout (Oncorhynchus mykiss)

Beth M. Cleveland a,⁎, P. Brett Kenney b, Meghan L. Manor b, Gregory M. Weber a a National Center for Cool and Cold Water Aquaculture, ARS/USDA, 11861 Leetown Rd, Kearneysville, WV 25430, United States b Division of Animal and Nutritional Sciences, Davis College of Agriculture, Forestry, and Consumer Sciences, West Virginia University, PO Box 6108, Morgantown, WV 26505, United States article info abstract

Article history: Sexual maturation in salmonids requires mobilization of proteins from muscle tissue, which is evidenced by Received 10 August 2011 increased expression of proteolysis-related genes and decreased muscle protein content. However, it is un- Received in revised form 21 December 2011 known how ration level affects this proteolytic response. In the current study, female diploid rainbow Accepted 30 January 2012 trout (Oncorhynchus mykiss) approaching ovulation were fed for 12 weeks at 0.25% and 0.50% tank biomass, Available online 12 February 2012 and to apparent satiation. Triploid trout, which exhibit little ovarian growth, were included at the 0.50% ration level. Gonad somatic index increased in diploids from 3.98% to approximately 13% and was unaffected Keywords: Fish by ration. Reduced feed intake and maturation negatively affected protein and lipid deposition in skeletal Spawning muscle, as indicated by reduced fillet weight and reduced fillet protein and lipid content. During mid- Protein turnover vitellogenesis, expression of genes involved in proteolytic pathways was higher in diploids compared to trip- GeXP loids and the majority of the differentially expressed transcripts were cathepsin and autophagy-related Atrogin genes. These differences increased with maturation and expanded to include multiple components of the proteasome, ubiquitin ligases, calpastatins, and caspase 9. Expression patterns of multiple proteolysis- related genes suggest that fish consuming the moderate 0.50% ration had the lowest capacity for protein deg- radation. In summary, these results suggest that maturation increases protein degradation and that higher levels of feed intake are unable to alleviate these effects. However, higher levels of feed intake prevent a net loss of muscle protein, suggesting that dietary nutrients are able to replace endogenous nutrients mobi- lized from skeletal muscle in support of gonad growth. Published by Elsevier B.V.

1. Introduction tissues can be mobilized to sustain vitellogenesis and oocyte growth. Therefore, vitellogenesis and oocyte growth are relatively unaffected In female rainbow trout, sexual maturation can increase ovarian by changes in nutrient supply, with dietary energy or nutrient defi- weight to represent greater than 20% of body weight prior to ovula- ciencies overcome at the expense of endogenous energy and nutrient tion (Tyler et al., 1990). An overwhelming proportion (80–90%) of stores (Nassour and Leger, 1989). dry egg mass is composed of yolk proteins, which are derived from During maturation, endogenous lipids are preferentially mobilized vitellogenins and serve as a nutrient source for the developing em- from visceral stores, with additional lipids mobilized from muscle bryo (Finn and Fyhn, 2010). Therefore, during the vitellogenic stage tissue when necessary (Aussanasuwannakul et al., 2011; Jonsson of oocyte development, the rate of ovary growth is primarily a func- et al., 1997; Nassour and Leger, 1989; Shearer, 1994). In contrast, tion of the rate at which vitellogenin is deposited in the oocyte protein is mobilized primarily from muscle (Jonsson et al., 1997)to (Lubzens et al., 2010). Vitellogenins are synthesized in the liver, yield amino acids, which can be oxidized into energy or used as pre- driven by increased levels of plasma 17β-estradiol (E2) (Davis et al., cursors for vitellogenin synthesis and oocyte development. Indices of 2009; Schafhauser-Smith and Benfey, 2003) produced by the somatic protein degradation increase in white muscle of maturing salmonids, cells of the developing ovarian follicle. which are observed as an up-regulation of cathepsin activity (Ando Although the nutritional plane of the fish throughout maturation et al., 1986; Mommsen, 2004; Salem et al., 2006b) and increased affects egg size, it does not affect the proximate composition of proteolysis-related gene expression across multiple pathways of pro- the egg (Knox et al., 1988; Ridelman et al., 1984; Washburn et al., tein degradation (Salem et al., 2006a; Salem et al., 2006b; Wang et al., 1990), indicating that the energy and nutrients stored in somatic 2011). However, it is still unclear whether increased muscle prote- olysis in maturing females is induced by nutrient insufficiencies or hormonal signals associated with maturation. Although nutrient ⁎ Corresponding author at: NCCCWA, 11861 Leetown Rd, Kearneysville, WV 25430, United States. Tel.: +1 304 724 8340x2133; fax: +1 304 725 0351. deprivation up-regulates proteolytic processes in white muscle E-mail address: [email protected] (B.M. Cleveland). (Cleveland and Evenhuis, 2010; Loughna and Goldspink, 1984;

0044-8486/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.aquaculture.2012.01.032 B.M. Cleveland et al. / Aquaculture 338–341 (2012) 228–236 229

Rescan et al., 2007), effects of sex steroids on protein degradation in family C, 8 of the 28 fish were males, and in family D 13 of the 28 fish fish remain unclear. Salmonid research indicates that exogenously were males. Since there were not enough females in family D to allow administered E2 promotes protein catabolism in muscle (Nazar for sampling, data from this family were not included in the study for et al., 1991; Olin et al., 1991; Toyohara et al., 1998). Therefore, eleva- either sampling date, although they remained in the tanks. Only data tions in circulating E2 during maturation may mobilize muscle amino from female fish were included in the analysis. acids via protein degradation, regardless of whether nutrient intake Fish were fed Zeigler G floating 5.0 mm (3/16") pelleted feed (42% is sufficient to support gonadal development. protein, 16% fat, 2% fiber; Zeigler Brothers, Inc.; Gardners, PA) dis- The current study investigates effects of sexual maturation and ra- pensed by automatic feeders (Arvotec, Huutokoski, Finland) that tion level on carcass characteristics, as well as proteolysis-related adjust feeding daily based on the predicted mass of the fish in the gene expression and plasma 3-methylhistidine as indices of protein tank. Feeders dispensed feed in multiple feeding events between degradation in female rainbow trout. Effects of maturation were 7 am and 2 pm. Fish from each tank were weighed monthly to main- determined by comparing maturing diploid females (2N, two sets of tain the accuracy of the feeder system. Feeders for those tanks fed to chromosomes) to sterile triploid female fish (3N, three sets of chro- satiation dispensed feed at 0.50% of tank biomass/day, followed by mosomes); the latter exhibit little gonad growth and E2 production hand-feeding at the end of day to apparent satiation. Feeding proce- (Nakamura et al., 1987). Additionally, multiple ration levels were dures were modified starting December 1 to reduce the number of applied in the experimental design to determine if increasing nutrient feeding events, which reduces competition for available feed by in- availability alleviates the effects of maturation on protein degrada- creasing the amount of feed provided per feeding. This promotes a tion. Proximate analyses indicate the crude nutrient profile of a tissue more even feed consumption among individuals, especially in those or body system at a given time point. These data, in combination tanks assigned to the lower ration levels. This was achieved by with measures of indices of protein degradation, are reflective of the placing collection buckets under the feeders for the tanks receiving effects of maturation and feeding level on protein turnover rates in the 0.25% and 0.50% rations to collect the dispensed feed. The collect- skeletal muscle. ed feed was then hand-fed to the fish at 8 am the next day, with a sec- ond feeding at 2 pm if during the first feeding event the fish reached 2. Methods and materials satiation before all the collected feed was dispensed.

2.1. Experimental design 2.2. Sample collection

Diploid and triploid rainbow trout were generated from each of Fish were harvested using an overdose of tricaine methanesulfo- 4 families (families A, B, C, and D), and were confirmed as diploid or nate (MS-222, Western Chemicals, Ferndale, CA) at 300 mg/L. Body triploid by flow cytometry (Allen, 1983; Hershberger and Hostuttler, weights and lengths were recorded. A 1 mL blood sample was re- 2007). At the fingerling stage, the fish were implanted with passive moved from caudal vasculature and stored on ice until plasma was integrated transponders (Avid Identification Systems, Inc.; Norco, separated by centrifugation as described in Section 2.5. Gonads CA) in the dorsal musculature for individual identification. At this were removed from the carcass, weighed, and a subsample of 25 fol- same time up to 14 diploid fish per family were combined into each licles was stored in 70% ethanol for measurement of follicle diameter. of 2 1000 L tanks and up to 14 triploid fish per family were combined Empty gastrointestinal tract weight, liver weight, and eviscerated into a separate set of 2 1000 L tanks. The diploid and triploid fish were body weight were also recorded. At the end of the study skinless, reared in separate tanks because communal rearing of triploid and boneless fillets were cut from each fish and weighed, and individual diploid rainbow trout often results in decreased growth of the triploid muscle subsamples were retained from the dorsal region of each animals (Piferrer et al., 2009). Fish were maintained indoors under fillet and frozen at −20 °C for analysis of proximate composition as simulated ambient photoperiod, and supplied with partially recircu- described in Section 2.3. A second dorsal muscle subsample was lated treated spring and well water throughout the study. Water immediately frozen in liquid nitrogen and stored at −80 °C for gene temperatures ranged from 14.0 °C in October to 12.4 °C in January. expression analysis. One month prior to the onset of the study, fish were fed at 0.75% of tank biomass/day. On November 3, 2 fish per family were sampled 2.3. Proximate analyses from each of the four tanks. At this same time, all fish were weighed, fork length recorded, and fish were transferred into 1000 L experi- Boneless, skinless fillets were ground while still frozen using a mental tanks. The initial ration levels for diploids were 1) apparent Kessel table-top meat grinder. A sub-sample of the ground muscle satiation, 2) 0.75% of tank biomass/day, and 3) 0.50% of tank was frozen in liquid nitrogen, powdered using a Waring commercial biomass/day, with triploids fed at 0.75% of tank biomass/day. Two grade blender, and stored at −20 °C until analysis. Moisture, protein, tanks were assigned to each of the four treatments, with a total of 7 and lipid analysis were completed using AOAC approved methods fish per family per treatment. Each family was split between two (AOAC, 2000). Moisture content was performed by weighing the tanks, with the first tank containing 4 fish from each family A and B, sample before and after an 18 h drying period at 110 °C. Crude pro- and 3 fish from each family C and D. The second tank contained 3 tein was determined from Kjeldahl N analysis while lipid content fish from each family A and B, and 4 fish from each family C and D. was determined using Soxhlet extraction methodology. Therefore, each tank contained an equal number of fish (n=14). Two weeks into the 12 week study, it was calculated that the fish 2.4. Multiplex gene expression analysis fed to satiation were consuming feed equivalent to a ration of 0.80– 0.90% of tank biomass/day. Therefore, ration levels were adjusted The GenomeLab GeXP genetic analysis system (Beckman Coulter, to 1) apparent satiation, 2) 0.50% of tank biomass/day and, 3) 0.25% Inc.) was used to simultaneously analyze expression of twenty- tank biomass/day for the remaining 10 weeks to increase the distance seven genes in white muscle tissue. Within the multiplex, twenty- between the satiation and the next-lowest ration level. The ration three genes were associated with proteolytic pathways and four level for the triploid fish was also decreased from 0.75% tank bio- served as potential reference genes. Primers were designed using mass/day to 0.50% tank biomass/day. The second and final sampling eXpress Designer software (Beckman Counter, Inc.) and primer se- period occurred on January 26. During both sampling periods, fish quences were compared against other rainbow trout gene sequences were selected for sampling based on tag number. Although all diploid using the BLAST function within the NCBI database to reduce unin- fish were expected to be female, males were found in two families. In tended sequence amplification. The size of each amplicon was 230 B.M. Cleveland et al. / Aquaculture 338–341 (2012) 228–236 confirmed with its expected length. No undetermined peaks inter- solution and 0.5 μL size standard 400. The PCR products were separat- fered with amplification of the intended multiplex. Optimization of ed by capillary electrophoresis in the GeXP Genetic Analysis System the multiplex, standard curve, reverse transcriptase (RT) and PCR re- using the Frag-3 protocol. actions, and capillary electrophoresis were performed as recom- Areas for each peak within the multiplex were exported to eX- mended by the manufacturer (GeXP Chemistry protocol A29143AC; press Profiler software (Beckman Coulter, Inc.) for analysis and nor- February, 2009) with reagents provided in the GeXP Start Kit (Beck- malization to the internal kanamycin control. Concentrations were man Coulter, Inc.). GenBank accession numbers for the sequences interpolated from the standard curves for each gene of interest. used to generate multiplex primers, their associated proteolytic path- Data were normalized to the highest expressing sample for input way, and R2 values for the RNA standard curve (0.2 ng/μL–10 ng/μL), into GeNorm software to determine which reference genes were are shown in Table 1. Primer sequences and expected amplicon size most stable. The most stable reference genes were actb, rplp2, and have been previously published (Cleveland and Weber, 2011). eef1a, with M-values of 0.456, 0.451, 0.345, respectively, therefore To isolate RNA, 50–100 mg of white muscle was homogenized in their geometric mean was used to generate a normalization factor 1 mL TRIzol (Invitrogen, Carlsbad, CA) per manufacturer's suggested for each sample. Thus, the normalized expression of each gene tran- protocol using a 5 mm steel bead and a multi-tube shaker. The RNA script is reported as the quantity relative to the geometric mean of pellet was washed twice with 75% ethanol, and resuspended in the selected reference genes. nuclease-free water. RNA quality and quantity was determined by measuring absorbance at 260 nm and 280 nm. For the RT reaction, 2.5. 3-Methylhistidine analysis 100 ng of DNase treated RNA was diluted 1:20 with nuclease-free water, and 2.5 μL was used in a 10 μL RT reaction that included 2 μL Plasma levels of 3-methylhistidine (3-MH) were determined 5× RT buffer, 1 μL gene-specific reverse primer mix, 0.5 μL RT, and using a previously published high pressure liquid chromatography 2.5 μL kanamycin RNA (internal control, 1:8 dilution). The RT was (HPLC) with electrochemical detection procedure (Cleveland et al., incubated according to kit instructions. An aliquot (4.65 μL) of the re- 2009). Briefly, plasma was deproteinized by vortexing 100 μL plasma sultant cDNA was used in a PCR reaction that included 2 μL25mM with 350 μL ice-cold methanol and incubating the mixture on ice for

MgCl2,2μL 5× PCR buffer, 1 μL forward primer mix, and 0.35 μL 30 min. After centrifugation at 15,000 g for 15 min (4 °C), the super- DNA Taq polymerase. The PCR was incubated according to kit instruc- natant, containing deproteinized plasma, was removed and stored tions. The PCR products were diluted 1:4 with 10 mM Tris–HCl at −20 °C until derivitization of individual samples immediately and 1 μL of the dilution was combined with 38.5 μL sample loading before injection onto the column. Plasma was mixed with an equal volume of derivitization solution (2 mM o-phthaldialdehyde and 0.04% β-mercaptoethanol in 0.1 M sodium tetraborate), incubated at Table 1 room temperature for 2 min, and injected (20 μL) onto a C18 column Genes included in multiplex and their associated function. (5 μm, 4.6×150 mm, Acclaim 120, Dionex, Sunnyvale, CA) with a 4.6×10 mm guard column attached to an HPLC system (pump Gene Gene name NCBI gene Standard curve symbol accession no. R2 value model p680, Dionex) with a manual Rheodyne injector and electro- chemical detector (model ED50, Dionex). 3-Methylhistidine was Reference genes rplp2 Acidic ribosomal protein P2 BT074359 0.9985 eluted from the column using an elution gradient that gradually actb Beta-actin NM_001124235 0.9930 increased the percentage of eluent B through the column at a flow gapdh Glyceraldyhyde phosphate NM_001124246 0.9923 rate of 1.2 mL/min. Eluent A contained 12.5 mM phosphate buffer dehydrogenase (pH 7.0) with 5 mL tetrahydrofuran per liter. Eluent B contained eef1a Elongation factor 1-alpha NM_001124339 0.9928 53% 12.5 mM phosphate buffer (pH 7.0), 40% acetonitrile, and 7% Ubiquitin–proteasome fi psma5 Proteasome alpha subunit-type 5 BX078765 0.9962 tetrahydrofuran. All eluents were vacuum ltered and degassed by psmb3 Proteasome beta subunit-type 3 NM_001124250 0.9924 sonification before use. The electrochemical detector was set at a psmd6 26S proteasome non-ATPase NM_001165054 0.9961 10.0 Hz data collection rate and 0.70 V direct current. regulatory subunit 6 ub Polyubiquitin NM_001124306 0.9986 ube2n Ubiquitin conjugating enzyme NM_001160651 0.9974 2.6. Statistical analysis fbxo32 F-box protein-32/atrogin-1 HM189693 0.9952 fbxo25 F-box protein-25 NM_001193325 0.9976 Data were analyzed using analysis of variance to test for main ef- murf1 Muscle RING finger protein-1 HM357611 0.9950 fects of ration level and family using PC-SAS (Version 9.1) general lin- murf2 Muscle RING finger protein-2 HM357612 0.9655 ear models procedure. Effects were considered significant at P≤0.05. murf3 Muscle RING finger protein-3 HM357613 0.9884 fi Cathepsin–lysosome When signi cant, differences between ration level were determined ctsd Cathepsin D NM_001124711 0.9994 using Fischer's least significant difference (LSD) procedure. Differ- ctsl Cathepsin L NM_001124305 0.9987 ences between diploid and triploid fish at the 0.50% ration level, atg4b Autophagy-related protein BX914166 0.9980 and mature and immature fish at the 0.25% ration level were detected 4 homolog B fi atg12 Autophagy-related protein 12 CB490089 0.9939 with a t-test analysis. Differences were considered signi cant lc3b Microtubule-associated CA350545 0.9950 at P≤0.05. To normalize gene expression data, fold change values proteins 1A/1B light chain 3B were log2 transformed prior to statistical analysis. Data are presented gabarapl1 Gamma-aminobutyric acid CA345480 0.9910 as means±SEM (standard error of the mean). receptor associated protein Calpain system capn1 Calpain-1 catalytic subunit NM_001124490 0.9942 3. Results (micro) capn2 Calpain-2 catalytic subunit NM_001124491 0.9876 3.1. Feed intake (milli) castl Calpastatin-long isoform NM_001124538 0.9965 fi casts Calpastatin-short isoform NM_001124645 0.9961 The ration for sh consuming feed to satiation was calculated Caspases to average 0.68% of tank biomass/day. Therefore, fish assigned to casp3 Caspase-3 CA366923 0.9979 the 0.50% ration level were estimated to consume feed at approxi- casp8 Caspase-8 CU072654 0.9858 mately 74% of satiation; consequently they were moderately feed casp9 Caspase-9 NM_001124647 0.9966 restricted. Fish at the 0.25% ration level were consuming feed at B.M. Cleveland et al. / Aquaculture 338–341 (2012) 228–236 231

Table 2 maturation or ration level was not dependent on family association. Carcass characteristics and organ indices for diploid and triploid fish harvested at the Means were therefore pooled across all families within treatment, and beginning of the study. Values are means±SEM. only the main effects of ration level and sexual maturation are discussed. Diploid Triploid fi Carcass characteristics 3.4. Carcass and llet characteristics ⁎ Body wt, g 1351 ±58 1015 ±50 ⁎ Eviscerated body wt, g 1160 ±49 901±42 3.4.1. Effect of sexual maturation Empty gastrointestinal tract wt, g 76.8±7.2 73.5±5.3 ⁎ Differences in carcass characteristics were present between dip- Gonad wt, g 55.0±7.3 0.49±0.07 ⁎ Liver wt, g 23.3±1.3 13.8±1.1 loid and triploid females at the beginning of the study (Table 2). ⁎ Length, mm 446±6 410±9 Body weight, eviscerated body weight, gonad weight, and GSI were Condition factor 1.51±0.03 1.47±0.02 greater in diploid fish compared to triploid fish. A GSI of 3.98 and Egg diameter, mm 2.85±0.09 – mean follicle diameter of 2.85 mm in the maturing diploids indicate they were at mid-vitellogenesis (Lankford and Weber, 2010). The Organ index ⁎ HSI 1.72±0.04 1.35±0.06 hepatosomatic index (HSI) of diploids was also greater than that ⁎ GSI 3.98±0.48 0.048 ±0.003 of triploids, whereas the gastrointestinal tract somatic index (GtSI; ⁎ GtSI 5.72±0.39 7.21±0.30 [weight of empty gastrointestinal tract/total body wt×100]) was n1110greater in triploids versus diploids. ⁎ Asterisks represent a significant difference (Pb0.05) between diploid and triploid fish. The effects of ploidy and maturation on carcass and fillet charac- teristics at the end of the study are shown in Table 3. Effects of ploidy approximately 37% of satiation, and therefore are considered a more and maturation can be determined by comparing triploid and mature extreme case of nutrient restriction. diploid fish consuming the 0.50% ration level (triploid—0.50% vs diploid—0.50%) as well as immature and mature diploid fish at the 3.2. Immature diploids 0.25% ration level. Consistent with what was observed at the begin- ning of the study, triploid fish remained smaller than diploids con- Two diploid fish at the 0.25% ration level and one diploid at the suming the same ration. Within the 0.25% ration, immature diploid 0.50% ration failed to mature. These three fish were identified as fish gained more weight than maturing fish. Gastrointestinal tract having undeveloped gonads and were members of the same family. weight and GtSI were significantly higher in immature diploids and Data from the two immature diploid fish at the 0.25% ration level triploids compared to diploid fish at similar levels of feed intake. Con- were compared against mature fish within the same ration level to dition factor was greater in mature diploid fish versus triploid fish at detect main effects of sexual maturation. The single immature diploid the 0.50% ration level. The GSI for mature diploids increased by about fish at the 0.50% ration level was not used in the analysis. 3-fold during the study and gonad weight represented over 12% of body weight; while triploids and immature diploids did not exhibit 3.3. Family effects gonad development and had GSI values less than 0.15%. Mature diploids had greater HSI values than triploids, but not immature Although main effects of family were observed for several variables diploids. Skinless fillet weight, as a percentage of eviscerated body of carcass characteristics and gene expression, when the data for imma- weight, was greater in immature diploids compared to sterile dip- ture fish are removed there were no notable family by ration or family loids. Percent fillet lipid and protein were lower in mature fish than by ploidy interactions. Therefore, data variation explained by immature fish at the 0.25% ration level. However, percent fillet lipid

Table 3 Effects of maturation and ration level of carcass and fillet characteristic. Values are means±SEM. Means without a common letter represent significant differences between mature diploids, Pb0.05.

Mature diploid Immature diploid Triploid

0.25% 0.50% Satiation 0.25% 0.50%

Carcass characteristics b a a ⁎ Body wt, g 1582±63 1890±57 2005±127 1624 ±217 1343±60 b a a ⁎ Eviscerated body wt, g 1250±44 1507±51 1582±100 1400 ±169 1179±51 b a a ‡ ⁎ Empty gastrointestinal Tract wt, g 53.7±5.1 74.8±4.5 69.3±5.6 160.2±18.0 109.1±5.6 b a a ⁎ Fillet wt, g 644±35 825±27 854±33 852±40 623±31 b ab a ‡ ⁎ Gonad wt, g 217±21 234±16 272±16 2.55±0.12 0.55±0.07 b a ab ⁎ Liver wt, g 25.6±2.5 36.2±1.6 31.2±3.5 20.1±1.0 16.8±1.2 ⁎ Length, mm 461±7 477±6 483±9 446±15 449±7 b a a ⁎ Condition factor 1.61±0.03 1.74±0.06 1.75±0.03 1.58±0.05 1.48±0.06 c b a ‡ ⁎ Body wt gain, % 12.2±1.4 34.7±1.8 45.4±4.5 19.2±1.0 25.7±2.3 Egg diameter, mm 4.38±0.08b 4.60±0.06a 4.62±0.07a ––

Organ index, g/100 g b a b ⁎ HSI 1.60±0.13 1.92±0.07 1.58±0.16 1.30±0.09 1.24±0.04 ‡ ⁎ GSI 13.7±0.9 12.4±0.7 13.7±0.9 0.15±0.02 0.04±0.01 ‡ ⁎ GtSI 3.36±0.30 3.97±0.24 3.45±0.22 9.20±1.31 8.08±0.23

Fillet composition, % b a a ‡ ⁎ Fillet yield 51.4±0.6 54.8±0.5 53.9±0.6 57.1±0.3 52.8±0.5 Moisture 75.8±0.4a 73.3±0.4b 73.1±0.4b 73.0±0.3‡ 72.4±0.3 Protein 19.2±0.2b 20.0±0.2a 19.6±0.2ab 20.5±0.3‡ 20.0±0.3 Lipid 5.11±0.47b 6.67±0.47a 7.28±0.34a 8.05±0.46‡ 7.25±0.32 n 12 9 11 2 10

⁎ Asterisks represent a significant difference (Pb0.05) between mature diploid and triploid fish at the 0.50% ration level. ‡ Double daggers represent a significant difference (Pb0.05) between mature and immature diploid fish at the 0.25% ration level. 232 B.M. Cleveland et al. / Aquaculture 338–341 (2012) 228–236 and protein were not different between mature diploid and immature involved in the cathepsin–lysosome proteolytic pathway were all dif- triploid fish at the 0.50% ration level. ferentially expressed between diploid and triploid fish (Fig. 1C and D). At the end of the study, the frequency of differentially expressed 3.4.2. Effect of ration level genes between mature and non-maturing fish increased and differ- Effect of ration level on carcass characteristics and fillet quality ences were observed across all proteolytic systems (Fig. 2). Three in mature diploid trout are shown in Table 3. Body weight, gastroin- subunits of the proteasome, psma5, psmb3, and psmd6, exhibited the testinal tract weight, fillet weight, condition factor, percent weight lowest expression in triploid and immature diploid fish (Fig. 2A) gain, and follicle diameter were all lower in the fish at the 0.25% compared to mature diploid fish at the 0.50% and 0.25% ration levels, ration level compared to the two higher ration levels. Additionally, respectively. Expression of ubiquitin ligase genes fbxo32 (atrogin-1), with the exception of percent weight gain, increasing feed intake fbxo25, murf2, and murf3 (Fig. 2B) were lower in immature fish com- from 0.50% tank biomass/day to satiation did not significantly alter pared to mature fish consuming similar ration levels (Fig. 2B). Across the aforementioned carcass characteristics. While GSI and GtSI were the cathepsin–lysosome pathway, cathepsin genes ctsd and ctsl,as not affected by ration level, fish consuming the 0.50% ration exhibited well as three genes (atg4b, lc3b, gabarapl1) involved in autophagic greater HSI than fish consuming 0.25% and satiation ration levels. processes were higher in maturing diploids compared to triploids There was a significant effect of ration level on fillet yield and pro- and immature diploids (Fig. 2C–D). Although neither of the calpain ximate composition; fish consuming the lowest ration exhibited genes (capn1 and capn2) were differentially expressed, the inhibitory lower percent fillet yield and lipid content, and greater moisture calpastatins, castl and casts, exhibited lower expression in immature content than fish consuming the two higher rations, and lower pro- diploid and triploid fish compared to maturing fish at the same ration tein content than fish at the moderate ration. level (Fig. 2E). Among the three caspase genes in the multiplex, casp9 remained the only one to be affected by maturation, with higher 3.5. Gene expression expression in mature fish compared to immature diploid and triploid fish within the same ration level (Fig. 2F). 3.5.1. Effect of sexual maturation At the beginning of the study, ten of the twenty-three proteolysis- 3.5.2. Effect of ration level related genes were expressed at a higher level in diploid versus triploid The three proteasome subunits (psma5, psmb3, and psmd6) and fish (Fig. 1A–F). The six genes included in the multiplex that are the ubiquitin conjugating enzyme, ube2n, responded to ration level

0.8 0.8 A B 2N 3N 0.6 * 0.6

0.4 0.4

Fold ChangeFold 0.2 * 0.2

0.0 0.0 psma5 psmb3 psmd6 ub ube2n fbxo32 fbxo25 murf1 murf2 murf3

0.4 CD0.8

0.3 0.6 *

0.2 * * 0.4

Fold Change * * 0.1 0.2 *

0.0 0.0 ctsd ctsl atg4b atg12 lc3b gabarapl1

0.8 EF0.6 0.5 0.6 0.4 * * 0.4 0.3

0.2 Fold Change 0.2 0.1

0.0 0.0 capn1 capn2 castl casts casp3 casp8 casp9

Fig. 1. Differences in proteolysis-related gene expression in diploid and triploid fish at the beginning of the study. Panels represent genes associated with A) the ubiquitin–protea- some pathway, B) ubiquitin ligases, C) cathepsins, D) autophagy-related genes, E) the calpain pathway, and F) caspases. Values are means±SEM and represent the fold change in gene abundance, relative to the normalized mean of three reference genes (actb, rplp2, and eef1a). Asterisks represent a significant difference between diploid (2N) and triploid (3N) fish, Pb0.05. B.M. Cleveland et al. / Aquaculture 338–341 (2012) 228–236 233

2N-0.25% 2N-satiation 3N-0.50% 2N-0.50% 2N-immature

0.8 0.7 A a B a 0.6 a ‡ ab 0.6 a ab a b b 0.5 a b a b 0.4 ‡ 0.4 ‡ ab b * ‡ * b b 0.3 * b ‡ ‡ Fold Change 0.2 0.2 ‡ * * 0.1 * ‡ 0.0 0.0 * psma5 psmb3 psmd6 ub ube2n fbxo32 fbxo25 murf1 murf2 murf3

0.6 0.8 CDa

a 0.5 ab 0.6 a a 0.4 ab b b 0.3 b 0.4 ‡ * 0.2 Fold Change ‡ ‡ ‡ * 0.2 * 0.1 * ‡ *

0.0 0.0 ctsd ctsl atg4b atg12 lc3b gabarapl1 0.8 EF0.8

0.6 0.6 a

a

0.4 ‡ 0.4 * b Fold Change ‡ 0.2 ‡ * 0.2 *

0.0 0.0 capn1 capn2 castl casts casp3 casp8 casp9

Fig. 2. Effects of maturation and ration level on proteolysis-related gene expression in fish at the end of the study. Panels represent genes associated with A) the ubiquitin–protea- some pathway, B) ubiquitin ligases, C) cathepsins, D) autophagy-related genes, E) the calpain pathway, and F) caspases. Values are means±SEM and represent the fold change in gene abundance, relative to the normalized mean of three reference genes (actb, rplp2, and eef1a). Means without a common letter represent significant differences among mature diploid (2N) fish, Pb0.05. Asterisks (*) represent a significant difference (Pb0.05) between mature diploid and triploid (3N) fish at the 0.50% ration level and are shown over the bars for the triploid fish. Double daggers (‡) represent a significant difference between mature and immature diploid fish at the 0.25% ration level and are shown over the bars for the immature diploid fish.

(Fig. 2A). These genes consistently exhibited greatest expression MH concentrations in mature diploids were approximately twice levels at the 0.25% ration level and lowest expression levels at the that in immature diploids at the same ration level (Fig. 3B). There 0.50% ration level (Fig. 2A). In addition, two ubiquitin ligase genes, was no difference in plasma 3-MH concentrations between mature murf2 and murf3 (Fig. 2B), the cathepsin genes ctsd and ctsl diploid fish and triploid fish at the 0.50% ration level. (Fig. 2C), and the autophagic and calpastatin genes, lc3b and casts (Fig. 2D–E), all responded to ration level in a similar manner; the greatest expression levels occurred at the 0.25% ration level and the 7 2N 7 2N-0.25% lowest expression levels were observed at the 0.50% ration level. A 3N B 2N-0.50% None of the three caspase genes responded to feed intake (Fig. 2F). 6 6 2N-satiation

M) 2N-immature 5 5 3N-0.50% 3.6. Plasma 3-methylhistidine 4 4 3 3 ‡ 3-Methylhistidine is a post-translationally methylated histidine residue found in myofibrillar proteins that, once liberated via degra- 2 2 dation, cannot be reincorporated back into proteins since they are Plasma 3-MH ( 1 1 unable to charge tRNA molecules. Therefore, changes in plasma 3- 0 0 MH are often used as an indicator of changes in rates of protein deg- First Sampling Final Sampling radation (Fruhbeck et al., 1996; Wassner et al., 1977). Fig. 3. Effects of maturation and ration level on plasma 3-methylhistidine (3-MH) con- centrations. Values are means±SEM. A) A significant difference was not observed 3.6.1. Effect of sexual maturation between diploid (2N) and triploid (3N) fish, P>0.05. B) Double daggers (‡) represent fi At the beginning of the study, diploid and triploid sh had similar a significant difference between mature and immature diploid fish at the 0.25% ration concentrations of plasma 3-MH (Fig. 3A). At the end of the study, 3- level. 234 B.M. Cleveland et al. / Aquaculture 338–341 (2012) 228–236

3.6.2. Effect of ration level combination of reduced rates of protein synthesis and increased Plasma 3-MH concentrations were not affected by feeding level rates of protein degradation are likely responsible for negative effects (Fig. 3B). of sexual maturation on protein accretion in fish. Driving this response may be increased E2 production, which promotes protein 4. Discussion catabolism and increases proteolysis-related gene expression in rain- bow trout primary myocyte cultures (Cleveland and Weber, 2011). Despite being on a relatively high nutritional plane, differences in Reduced feed intake during maturation may also reduce protein syn- proteolytic capacity were observed as differences in gene expression thesis; as there is a positive relationship between protein consump- between diploid and triploid fish at the beginning of the study; tion and protein synthesis in fish (Houlihan et al., 1995; Owen et al., when the diploid oocytes were at mid-vitellogenic growth. The 1999). majority of the differentially expressed proteolysis-related genes Previous research indicates that carcass protein content remains were centered within the cathepsin–lysosomal pathway, suggesting stable across different feeding levels (Bureau et al., 2006; Cleveland that any increase in protein degradation during this early period of and Burr, 2011). Therefore, the percent fillet yield and protein content maturation is predominately a function of increased activity of this of immature diploid fish at the 0.25% ration level at the end of the pathway. However, the general trend among genes associated with study are likely an accurate estimate of these variables in immature other proteolytic pathways was for triploid fish to exhibit numerical- diploid fish at the beginning of the study. Thus, average total muscle ly, albeit not significant, lower expression levels compared to diploid protein was calculated by multiplying eviscerated body weight by fish. Collectively, these observations suggest that rates of protein deg- percent fillet yield and percent fillet protein. Subtracting the amount radation between maturing diploids and triploids may be influenced of total muscle protein in fish at the end of the study from fish at the by multiple proteolytic pathways. beginning of the study estimates the amount of muscle protein Reductions in gastrointestinal tract weight and GtSI for maturing gained or lost throughout the study. Mature diploids consuming the diploids compared to triploids and immature diploids are consistent 0.25% ration lost approximately 12 g of muscle protein while imma- with previous literature, indicating that visceral lipid stores are mobi- ture diploids at the same ration level gained 28 g of muscle protein. lized during maturation (Aussanasuwannakul et al., 2011; Nassour Therefore, maturation induced a net loss of muscle protein in fish at and Leger, 1989). Additionally, reduced fillet lipid content in mature the lowest ration level. However, mature diploids consuming the fish compared to immature fish at the 0.25% ration level suggests mo- 0.50% and satiation rations gained approximately 29 g and 31 g of bilization of muscle lipid stores. The majority of these lipids are either muscle protein, respectively, indicating muscle atrophy is prevent- 1) oxidized for energy to meet the increased energy requirement for able with sufficient nutrition. Triploid fish at the 0.50% ration level vitellogenin production and gonad growth or 2) deposited in the gained 29 g of muscle protein, but due to differences in body weights oocyte as an energy source for the developing embryo. Previous re- a 29 g protein gain equals a 21% and 31% increase in muscle protein in search reports higher fillet lipid content in triploid fish compared to diploid and triploid fish, respectively. The 31 g protein gain in mature maturing diploid fish (Aussanasuwannakul et al., 2011). However, diploids at the satiation ration represents approximately a 23% pro- diploid and triploid fish were maintained in the same tank, which tein increase. Therefore, sufficient nutrition may only partially com- likely led to differences in feeding rate. Fillet lipid content was not pensate for the maturation-induced increases in protein degradation. significantly greater in triploids compared to maturing diploids at In the current study, regardless of ration level, mature fish gener- the same ration in the current study. This observation indicates that ally exhibit greater proteolysis-related gene expression than imma- at higher ration levels maturing diploids either did not mobilize lipids ture fish. These comparisons suggest that maturation-related from white muscle or that dietary nutrients were sufficient to replace signals, such as elevated E2, are regulators of protein degradation in lipids that were lost. muscle during sexual maturation. Thus, increases in protein degrada- Interestingly, although GtSI in maturing fish were considerably tion will occur during vitellogenesis, even during very high levels of lower than in immature or triploid fish, ration level did not affect feed intake and significant overall growth. It was anticipated that, as GtSI in mature diploid fish, suggesting that the mobilization of viscer- ration level increases, the reliance upon body stores for energy and al lipids may be regulated by maturation-related signals and not just nutrients would decrease, and progressively reduce the expression nutrient availability. Possibly, the increase in E2 that induces vitello- genes in protein degradation pathways. However, levels of gene ex- genesis also promotes the mobilization of proteins and lipids from pression were often at their numerical lowest in moderately restrict- muscle and visceral stores to fuel vitellogenin production. Other stud- ed fish at the 0.50% ration level, and a similar pattern as observed for ies have shown catabolic effects of E2 in fish (Cleveland and Weber, plasma 3-MH concentrations. This response pattern suggests that 2011; Schafhauser-Smith and Benfey, 2003; Toyohara et al., 1998). rates of protein degradation were at their lowest in moderately feed However, the reduced muscle lipid content for fish consuming the restricted fish. This conclusion is supported by a previous study in most limiting ration suggests that mobilization of muscle lipids is reg- juvenile rainbow trout (150–300 g) that indicate expression of ulated by the energy status of the fish. Therefore lipid mobilization proteolysis-related genes (fbxo32, murf, ctsd, and ctsl) and plasma 3- would occur when visceral and dietary lipid supplies are not suffi- MH were lowest when fish were moderately feed restricted at 60% cient for vitellogenesis. Previous research indicates that visceral of satiation (Cleveland and Burr, 2011). Additional studies have lipids are preferentially mobilized prior to lipids from somatic tissues shown that moderate levels of feed restriction correspond with slow- (Nassour and Leger, 1989), and the current study supports these est fractional rates of protein degradation (Houlihan et al., 1989), observations. maximum protein retention efficiency (Bureau et al., 2006), and re- Compared to their maturing counterparts, immature diploids ex- duced nitrogen output (Wang et al., 2007). Therefore, levels of feed hibit the greatest fillet yield and protein content, suggesting that pro- intake approaching satiation reduce protein retention, predominately tein accretion is negatively affected by maturation. Protein turnover via increased rates of protein degradation, prior to and during sexual may be a function of reduced rates of protein synthesis, increased maturation. Collectively, these findings suggest that moderate levels rates of protein degradation, or both. Rates of protein synthesis de- of feed restriction during vitellogenesis minimize the maturation- crease in salmon during sexual maturation (Arndt, 2000; Olin and induced increase in protein turnover without negative effects on von der Decken, 1987). The up-regulation of numerous genes that oocyte development. code for proteolytic proteins and increased plasma 3-MH concentra- Although casp9 increased only in response to maturation, genes tions supports previous research indicating an increase in protein within the three routes of protein degradation responded to matura- degradation during maturation (Salem et al., 2006b). Therefore, the tion and ration level. Effects were consistently of greatest magnitude B.M. Cleveland et al. / Aquaculture 338–341 (2012) 228–236 235 within the ubiquitin–proteasome and cathepsin pathways. Previously, negative effects on egg size. While satiation feeding promotes re- the cathepsin–lysosomal pathway has been implicated as primarily duced nutrient retention efficiency, lower rations reduce egg size responsible for muscle atrophy during maturation (Mommsen, and cause muscle loss, both with negative impacts on reproduction 2004; Toyohara et al., 1998; Yamashita and Konagaya, 1990); how- efficiency and profitability. This observation suggests moderate feed ever increased expression of proteasome subunits and murf3 (Wang restriction as an optimal feeding strategy for fish that are retained et al., 2011; Salem et al., 2006b) have also been reported. In addition for additional breeding cycles. to the aforementioned genes, the current study also associates ubiqui- tin ligase enzymes, fbxo32, fbxo25, and murf2, with maturation- induced muscle atrophy, as well as the autophagy-related genes Disclosure atg4b, lc3b, and gabarapl1. These later genes function in sequestering an encapsulated autophagosome containing cytosolic constituents Mention of trade names is solely for the purpose of providing and proteins to the lysosome for cathepsin-mediated degradation accurate information and should not imply product endorsement by (Levine and Yuan, 2005). Therefore, the increased expression of the United States Department of Agriculture. USDA is an equal oppor- autophagy-related genes that occurred during maturation is consis- tunity provider and employer. tent with an increased rate of protein degradation via the cathepsin– lysosomal pathway. Cathepsins are capable of degrading cytosolic Acknowledgements (Dice and Chiang, 1989), sarcolemmic (Delbarre-Ladrat et al., 2006), and myofibrillar proteins (Matsumoto et al., 1983; Schwartz and We thank Lisa Radler, Jill Birkett, and Mark Hostuttler for their Bird, 1977). Therefore the maturation-induced increase in cathepsin technical expertise. We acknowledge animal caretaking contributions expression may promote both selective and non-selective protein from Josh Kretzer, Jenea McGowan, Kyle Jenkins, and Kevin Melody. degradation. Funding for this study came from the Agricultural Research Service The most dramatic effect of maturation was observed for fbxo32 Project 1930-31000-010-000D and by Hatch funds (WVA00456) of expression, which increased greater than 20-fold during maturation. the West Virginia University, Agriculture and Forestry Experiment The Fbxo32 (also known as atrogin-1), Fbxo25, and Murf1-3 proteins, Station. are ubiquitin ligase enzymes that polyubiquitinate substrate proteins, subsequently inactivating and marking that protein for proteasomal degradation. In salmonids, fbxo32 and murf genes are negatively reg- References ulated by insulin-like growth factor-I (IGF-I) and insulin (Cleveland fi fi and Weber, 2010; Seiliez et al., 2011) and positively regulated by Allen, S.K., 1983. 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