The Influence of the Dietary Inclusion of the Alkaloid Gramine, on Rainbow Trout (Oncorhynchus Mykiss) Growth, Feed Utilisation and Gastrointestinal Histology

Aquaculture 253 (2006) 512–522 www.elsevier.com/locate/aqua-online

The influence of the dietary inclusion of the alkaloid gramine, on rainbow trout (Oncorhynchus mykiss) growth, feed utilisation and gastrointestinal histology

Brett Glencross a,d,*, David Evans a,d, Neil Rutherford a,d, Wayne Hawkins b,d, Peter McCafferty c,d, Ken Dods c,d, Brian Jones a, David Harris c,d, Lincoln Morton c,d, Mark Sweetingham b,d, Sofie Sipsas b,d

a Department of Fisheries-Research Division, PO Box 20, North Beach, WA 6020, Australia b Department of Agriculture-Government of Western Australia, Baron Hay Court, South Perth, WA 6150, Australia c Chemistry Centre (WA), 125 Hay St, East Perth, WA 6004, Australia d Centre for Legumes in Mediterranean Agriculture (CLIMA), Aquaculture Feed Grains Program, University of Western Australia, Crawley, WA 6909, Australia Received 9 March 2005; received in revised form 30 June 2005; accepted 1 July 2005

Abstract

This study examined the influence of the alkaloid gramine, when included in diets for rainbow trout. Quinolizidine alkaloids have been suggested as a potential anti-nutritional problem with the use of lupin (Lupinus sp.) meals in aquaculture diets. The findings from the present study show that above a critical threshold, the alkaloid gramine does have a strong anti-palatability effect. The effect is noted at a minimum gramine concentration of 500 mg/kg of diet, though not at 100 mg/kg. A continuing strong anti-palatability response is noted at higher inclusion levels and at the highest gramine inclusion concentration examined in this study (10,000 mg/kg), insufficient feed was consumed to even supply maintenance protein and energy demands. No adaptation to concentrations of gramine was observed throughout the 6-week study. No effects on nitrogen, energy or phosphorus digestibility were seen at the 500 mg/kg inclusion concentration of gramine relative to the reference diet, although the inclusion of the yellow lupin kernel meals (both Wodjil and Teo varieties) in the diet did improve the digestibility of phosphorus. Growth, as assessed using a range of parameters including weight gain, growth rate, nutrient and energy retention of fish fed the experiment treatments was largely consistent with feed intake. Survival of fish was significantly reduced at gramine inclusion levels above 1000 mg/kg. Feed conversion ratio (FCR) and feed conversion efficiency (FCE) were also reflective of feed intake and growth levels observed of each treatment. The concentrations of the plasma thyroid hormones tri-iodothyronine (T3) and thyroxine (T4) of fish from each of the treatments were consistent with feed intake (including the controls) suggesting that the concentrations of these hormones are in response to feed intake, not specifically the gramine levels in the diets. However, the inclusion of the Lupinus luteus kernel meals resulted in a significant change in T4 levels, with a degree of independence of the

* Corresponding author. Department of Fisheries-Research Division, PO Box 20, North Beach, WA 6020, Australia. Tel.: +61 8 9239 8103; fax: +61 8 9239 8105. E-mail address: [email protected] (B. Glencross).

0044-8486/$ - see front matter D 2005 Published by Elsevier B.V. doi:10.1016/j.aquaculture.2005.07.009 B. Glencross et al. / Aquaculture 253 (2006) 512–522 513 feed intake, suggesting that there may be another mechanism by which these meals are influencing the concentrations of this hormone. In this study, there was an increase in the density of melano-macrophage centres (MMC) with high dietary levels of gramine. However, in the absence of any histological evidence for a toxic effect, it is likely that the increased MMC densities observed in the fish fed high concentrations of gramine are associated with starvation. This study demonstrated that the lupin alkaloid gramine, can have a strong anti-nutritional effect on fish at inclusion concentrations greater than 100 mg/kg, but that its mode of action is primarily through an anti-palatability effect. It is therefore considered unlikely that alkaloid effects would be observed in diets even with 50% inclusion of kernel meals from Australian commercial L. luteus varieties. D 2005 Published by Elsevier B.V.

Keywords: Plant proteins; Fish meal replacement; Anti-nutritional factors; Alkaloids

1. Introduction Consumption of gramine at toxic levels in mice has been noted to lead to psychotropic levels of excite- It is well recognized in the aquaculture feeds indus- ment and seizure. The mode of action for gramine as try that there is a need to reduce reliance on fish meal an ANF, or toxicity data on this compound is limited. in aquaculture feeds (Naylor et al., 2000). Increasing However, mammalian effects include changes in the actual or prospective utilization of other protein tubules and glomeruli in the kidney, ureter and blad- meals in diets for aquatic species, substantial risk der, endocrine changes in spleen weight, and bio- reduction is achieved. The use of plant protein chemical changes such as enzyme inhibition, meals as alternative protein resources has been well induction via changes in blood or tissue levels of studied and many viable options including soybean, phosphatases (TXCYAC, 1980), although no specific glutens and lupin meals have been adopted indust- data is available for any fish species. Tolerance con- rially (Carter and Hauler, 2000; Storebakken et al., centrations to the inclusion of dietary gramine in other 2000; Glencross et al., 2004). However, the introduc- vertebrate species (rats, pigs and poultry) have been tion of anti-nutritional factors and other biologically determined at; about 300 mg/kg for rats, N500 mg/kg active compounds can accompany the use of plant diet for pigs and about 650 mg/kg diet for poultry protein meals (Francis et al., 2001). (Pastuszewska et al., 2001). The effects of concentra- Anti-nutritional factors (ANF) can affect the uti- tions as low as 250 mg/kg of Lupinus angustifolius lization of food by an animal through several ave- alkaloids have been reported in rats (Butler et al., nues, including the metabolic axis, nutrient 1996), although concentrations of alkaloids from L. digestibility or ingredient palatability (Refstie et al., albus were only reported to have an adverse effect at 1998, 1999; Glencross et al., 2003a,b). Alkaloids are 320 mg/kg (Zdunczyk et al., 1998). heterocyclic amino acid derivatives produced by The current Australian commercial L. luteus vari- plants as a chemical defence mechanism. While ety (Wodjil) has very low gramine concentration alkaloids are found in most legume species, they compared to European varieties such as Teo. How- have traditionally been found in high concentrations ever, Wodjil has proven agronomically costly to in the seeds of plants from the Lupinus genus (Pet- produce because of the high levels of insecticide terson et al., 1997; Wasilewko and Buraczewska, use required to deal with substantial insect infesta- 1999). Notably, a variety of alkaloids are found in tion problems (Perry et al., 1998; Berlandier and these seeds. In some varieties of the species Lupinus Sweetingham, 2003). There is evidence that aphid luteus, a major alkaloid component is gramine (Pet- infestation is directly related to the low inherent terson, 2000). Feeding studies with kernel meals from concentration of gramine (Risdall-Smith et al., the seeds of L. luteus have shown good prospect for 2004). Higher alkaloid varieties of L. luteus, such their use in aquaculture feeds because of their high as Teo, have better resistance to insect infestation, digestible protein content, although some deteriora- but it is unclear whether the higher alkaloids will tion in growth performance at high inclusion levels influence the usefulness of the kernel meal as an has been noted (Glencross et al., 2004). aquaculture feed ingredient. 514 B. Glencross et al. / Aquaculture 253 (2006) 512–522

This study reports on the nutritional influence of Table 1 gramine on the feed intake, growth, some biochemical Composition of the ingredients (all values are g/kg DM unless otherwise stated) parameters and tissue histology of rainbow trout. This c d e was examined over a range of inclusion concentra- Nutrient Fish Pregelled Cellulose Wodjil Teo meala wheat tions above and below naturally occurring concentra- starchb tions found in domesticated varieties of L. luteus. Dry matter 917 906 933 924 920 content (g/kg) Crude protein 770 7 3 512 541 2. Materials and methods Crude fat 68 11 2 79 79 Ash 142 3 2 54 73 2.1. Ingredients and diet preparation Crude fibre 0 10 660 33 35 Phosphorus 22 0 0 6 7 Organic matter 858 997 998 946 927 Purified gramine was purchased (Aldrich catalogue Gross energy 21.3 17.2 17.3 20.9 20.9 No. 1080-6, 99% purity). The gramine was dissolved (MJ/kg DM) in methanol and was added to a methanol-saturated Alkaloids 0 0 0 32 4087 cellulose slurry and the mixture was thoroughly (mg/kg DM) Arginine 43 0 0 47 61 mixed. The solvent was removed in vacuo and the Histidine 25 0 0 14 14 gramine/cellulose mixture was dried under vacuum. Isoleucine 28 2 0 17 20 Cellulose was used as a carrier for the gramine allow- Leucine 55 0 0 35 43 ing for easy dispersion of the gramine in the indivi- Lysine 46 1 0 23 17 dual diets. The gramine/cellulose mixture was added Methionine 21 0 0 4 3 Phenylalanine 29 0 0 18 21 to the experimental diets according to the formula- Threonine 32 2 0 16 19 tions in Table 1. All ingredients were ground such that Valine 34 0 0 17 19 they passed through a 600-Am screen. All experiment a Chilean Anchovy meal supplied by Skretting Australia, Cam- diets were formulated to be isonitrogenous (400 g/kg) bridge, Tasmania, Australia. and isoenergetic (19.5 MJ/kg) on a digestible nutrient b Supplied by Weston BioProducts, Henderson, Western Austra- lia, Australia. basis. Digestibility coefficient values for key ingredi- c ents were based on those reported earlier (Glencross et Supplied by ICN Biomedical, Costa Mesa, CA, USA. d Supplied by Coorow Seed Cleaners Pty Ltd, Coorow, Western al., 2005). Diets were processed by the addition of Australia, Australia. water (about 30% of mash dry weight) to all ingre- e Supplied by Department of Agriculture, South Perth, Western dients while mixing to form a dough. This dough was Australia, Australia. subsequently screw-pressed through a 3-mm diameter die using a pasta maker. The resultant moist pellets experiment, including dry matter, chromium, ash, were then oven dried at 70 8C for approximately 24 h fat, nitrogen, phosphorus and gross energy content. before being air-cooled, bagged and stored at 20 8C. Dry matter was calculated by gravimetric analysis The feed intake deterrent, sulfamerazine sodium was following oven drying at 105 8C for 24 h. Chromium added to two diets, based on the reference diet, at and phosphorus levels were determined using Induc- different levels to create a series of negative controls tively Coupled Plasma-Atomic Emission Spectro- (Boujard and Le Gouvello, 1997). Ingredient compo- scopy (ICP-AES) (McQuaker et al., 1979). Protein sition, diet formulations and diet composition are levels were calculated from the determination of presented in Tables 1–3 in that respective order. total nitrogen by Kjeldhal digestion, based on N6.25. Crude fat content was determined gravime- 2.2. Chemical analysis trically following extraction of the lipids according to the crude fat procedure (AOAC, 2000). Ash content All chemical analyses were contracted out to pro- was determined gravimetrically following loss of fessional chemical analytical laboratories. Respective mass after combustion of a sample in a muffle furnace samples of diet, faecal and whole-body samples were at 550 8C for 12 h. Organic matter content was analysed for a variety of analytes, depending on determined based on the difference between dry mat- B. Glencross et al. / Aquaculture 253 (2006) 512–522 515

Table 2 Formulations of the diets (all values are g/kg) Ingredient 0 10 100 500 1000 1500 10,000 Neg-1 Neg-2 Wodjil Teo Blend Chromium oxide 5 5 5 5 5 5 5 5 5 5 5 5 Pre-mix vitamins and mineralsa 5555555 5 5 5 55 Cellulose 113 112.9 112 108 103 98 13 108 103 22 30 26 Cellulose+100,000 mg/kg alkaloids 0 0.1 1 5 10 15 100 0 0 0 0 0 Pregelled starch 90 90 90 90 90 90 90 90 90 90 90 90 Fish oil 166 166 166 166 166 166 166 166 166 170 171 170.5 Fish meal 621 621 621 621 621 621 621 621 621 408 399 403.5 L. luteus cv. Wodjil kernel meal 0 0 0 0 0 0 0 0 0 300 0 150 L. luteus cv. Teo kernel meal 0 0 0 0 0 0 0 0 0 0 300 150 Sulfamerazine sodium 0 0 0 0 0 0 0 5 10 0 0 0 Source of ingredients provided in Table 1. a Supplied by Rhone Poulenc, Goodna, Queensland, Australia. Vitamin and mineral premix includes (IU/kg or g/kg of premix): Retinol, 2.5 MIU; Cholecalciferol, 0.25 MIU; a-tocopherol, 16.7 g; Menadione, 1.7 g; Thiamine, 2.5 g; Riboflavin, 4.2 g; Niacin, 25 g; Pantothenic acid, 8.3; Pyridoxine, 2.0 g; Folic acid, 0.8; Methylcobalamine, 0.005 g; Biotin, 0.17 g; Ascorbic acid, 75 g; Choline, 166.7 g; Inositol, 58.3 g; Ethoxyquin, 20.8 g; Copper, 2.5 g; Ferrous iron, 10.0 g; Magnesium, 16.6 g; Manganese, 15.0 g; Zinc, 25.0 g. ter content minus ash content. Gross energy was 0.58 g; meanFS.D.) hatchery reared rainbow trout determined by adiabatic bomb calorimetry. Concen- Oncorhynchus mykiss (Pemberton Strain; Molony et trations of tri-iodothyronine (T3) and thyroxine (T4) al., 2004). Treatments were randomly assigned in were determined by a competitive immunoassay quadruplicate to the tank array. Photoperiod was method using chemiluminescence detection (Fisher, maintained at 12L/12D. 1996). Gramine concentrations were determined by The fish were fed to apparent satiety once daily at extraction with trichloroacetic acid and then extracted about 0800 h for 42 days. Apparent satiety, as deter- from the aqueous layer with methylene chloride. The mined by a loss in feeding activity, was reached after gramine concentration was measured by gas chroma- three feeding sessions over a 1-h period. Uneaten feed tography using a capillary column (HP1, 30 m) and was removed from each tank 1 h later and the uneaten detected by a flame ionisation detector (Harris and portion dried and weighed to allow the determination Wilson, 1988). of daily feed intake based on correction factors for leaching losses sustained over an equivalent period. 2.3. Fish management Fish were individually re-weighed after 3 and 6 weeks, with all fish within each tank used to deter- Forty-eight shallow-conical bottomed 250 l tanks, mine the average weight gain per tank and treatment. with flow-through freshwater (4 l/min, salinity b1 Five fish were taken as an initial sample for composi- PSU and 14.1F0.8 8C, dissolved oxygen 9.7F0.3 tion analysis. At the end of the study, three fish were mg/l; meanFS.D., n =42), were each stocked with taken from each tank (4 replicates3 fish, per treat- 24, individually weighed, juvenile (9 month, 51.7 F ment) for whole body analysis. An additional three

Table 3 Composition of the diets (all values are g/kg DM unless otherwise stated) Ingredient 0 10 100 500 1000 1500 10,000 C1 C2 Wodjil Teo Blend Dry matter 960 954 945 951 949 959 966 950 951 958 950 957 Protein 419 426 418 424 416 429 417 426 426 448 436 447 Fat 231 231 232 228 227 228 230 228 227 203 217 209 Phosphorus 17 17 17 16 17 16 16 17 17 15 13 14 Ash 104 104 104 108 111 114 120 115 108 118 118 108 Gross Energy (MJ/kg DM) 22.88 23.44 23.17 23.25 23.12 23.35 23.52 23.24 23.41 23.16 23.48 23.53 Alkaloids (mg/kg) 0 10 116 547 1117 1867 11594 0 0 11 1179 512 516 B. Glencross et al. / Aquaculture 253 (2006) 512–522 fish from each tank were sampled for blood biochem- of the study and fixed in 10% neutral buffered for- istry, within 1 min of capture, by caudal tail vein malin. Incisions were made in the fish’s abdominal puncture using a 1-ml syringe fitted with at 20 G wall to allow penetration of the formalin. Following needle. Growth was assessed as mean weight gain preservation, the fish were dissected and samples of and daily growth coefficient (DGC). DGC was calcu- their liver, kidney, spleen, pyloric caeca and intestine lated as (Kaushik, 1998): were taken for histological examination. The samples were embedded in paraffin, sectioned at 5`m ı and 1=3 1=3 Wf Wi stained with haematoxylin and eosin using standard DGC ¼ 100: t techniques. A representative kidney section was stained with Perls stain for iron, Ziehl-Neelson for lipofuscin and Masson Fontana for melanin, using 2.4. Digestibility assessment standard techniques. The sample sections were examined for lesions. A At the end of the trial, faeces were collected using digital image (Olympus DP11) at 200 magnification stripping techniques based on those reported by Aus- was taken of each kidney sample and the density of treng (1978). Fish were netted from their respective melano-macrophage centres and pigment deposits in tank, placed in a smaller aerated tank containing AQI- the spleen were scored for each of the prints (1=few Sk (AQI-S NZ Ltd, Lower Hutt, New Zealand) (0.02 to 4=abundant). Scoring was performed without ml/l) until they lost consciousness. The faeces were access to the nutrition data, and repeated by three then removed from the distal intestine using gentle independent readers. abdominal pressure. Care was maintained to ensure that the faeces were not contaminated by urine and 2.6. Statistical analysis mucous. After removal of the faeces from the fish, the faecal sample was placed in a small plastic vial on ice All figures are meanFS.E. unless otherwise spe- and later stored in a freezer at 208C. Faeces were cified. Data were analysed for homogeneity of var- freeze dried prior to analysis. Sufficient faecal sample iances using Cochran’s test. Effects of diets were for analysis could not be obtained from some treat- examined by ANOVA using the software package ments, primarily because of low feed intake in some Statistica (StatsoftR, Tulsa, OK, USA). Levels of treatments. significance were determined using Tukey’s HSD Differences in the ratios of the parameters of protein test, with critical limits being set at P b0.05. Effects or gross energy to chromium, in the feed and faeces in of inclusion level of gramine on key performance each treatment were calculated to determine the appar- parameters were examined by linear and nonlinear ent digestibility coefficient (ADC ) for each of the diet regression modeling, also using the software package nutritional parameters examined in each diet based on Statistica. Variation between scorers for tissue histol- the following formula (Maynard and Loosli, 1969): ogy was examined using Friedman two-way ANOVA (SystatR, Richmond, CA, USA) and variation Crdiet Parameterfaeces ADCdiet ¼ 1 between trials was compared using Kruskal–Wallis Cr Parameter faeces diet analysis of variance (SystatR). where Crdiet and Crfaeces represent the chromium content of the diet and faeces, respectively, and Parame- terdiet and Parameterfaeces represent the nutritional 3. Results parameter of concern (protein or energy) content of the diet and faeces, respectively. 3.1. Influence of gramine on feed intake

2.5. Tissue histology One of the primary features noted with the increasing inclusion of gramine in the diet of the rainbow Two fish from each tank (n =24 per treatment) trout was the deterioration of feed intake with levels were euthanised with a sharp cranial blow at week 3 above 100 mg/kg DM (Table 4). The negative controls B. Glencross et al. / Aquaculture 253 (2006) 512–522 517

Table 4 Growth, feed intake, survival, utilisation efficiencies and hormonal levels of fish fed the experiment diets 0 10 100 500 1000 1500 10,000 C1 C2 Wodjil Teo Blend Pooled S.E.M. Initial weight (g/fish) 51.7a 51.9a 51.5a 51.6a 51.3a 52.0a 51.7a 52.0a 51.6a 51.6a 52.0a 51.3a 0.08 Final weight (g/fish) 172.7a 179.4a 178.8a 108.1c 59.9d 51.7de 45.3e 143.8b 119.7c 186.9a 61.8d 108.2a 7.61 DGC (%/day) 4.39a 4.55a 4.49a 2.47c 0.46d 0.02de 0.38e 3.58b 2.87c 4.74a 0.52d 2.49c 0.27 Gain (g/fish) 121.1a 127.5a 127.3a 56.6c 8.6d 0.3de 6.4e 91.8b 68.1c 135.2a 9.8d 56.9c 7.61 FCR (g feed: g gain) 0.83a 0.79a 0.80a 0.77a 2.61b 3.56c 1.04d 0.91a 0.96a 0.78a 2.75b 0.72a 0.45 FCE (g gain: g feed) 1.21ab 1.26a 1.24a 1.30a 0.38c 0.28c 0.96d 1.10ab 1.05b 1.28a 0.36c 1.40a 0.10 Feed intake 1.74a 1.68a 1.73a 1.14a 0.51c 0.49cd 0.32d 1.57ab 1.22b 1.76a 0.68c 1.09b 0.09 (g/fish/day)—week 1 Feed intake 3.12a 3.28a 3.27a 1.19d 0.28e 0.15e 0.09e 2.65b 1.96c 3.40a 0.23e 1.02d 0.20 (g/fish/day)—week 6 Feed intake 100.1a 101.1a 102.4a 43.1d 12.9e 11.4e 6.7e 83.4b 65.0c 105.6a 16.6e 40.6d 5.61 (g/fish)—total Survival (%) 100.0a 100.0a 98.6a 98.6a 94.4b 79.2d 86.1c 98.6a 98.6a 98.6a 83.3c 97.2ab 1.29 Nitrogen retention (%) 42.2a 42.1a 44.4a 44.6a 21.6b 0.4c 30.5d 40.7a 36.6a 44.3a 17.6b 44.1a 3.39 Energy retention (%) 60.1a 54.4a 54.6a 45.5b 11.0d 2.9e 32.0f 44.2b 39.6b 58.8a 8.2de 27.6c 4.43 a b a bc d de e ab c b cd bc Plasma free T3 (pmol/l) 9.6 8.1 10.6 7.1 4.0 3.5 3.1 9.0 6.7 8.3 5.1 7.4 0.44 a a a b bc c d b b bc b Plasma free T4 (pmol/l) 5.8 5.6 5.9 3.1 2.4 2.0 1.4 6.2 3.5 3.6 2.3 3.6 0.33

(C1 and C2) also had significantly poorer feed intake Palatability responses to the gramine diets were over the course of the experiment than the reference rapid and observed within a matter of days (Fig. 1). diet (no gramine, no sulferamerazine, no lupin diet) No adaptation to the gramine levels was observed and several of the lower level gramine inclusion diets. during the course of the experiment as was noted by Feed intake by fish fed the Wodjil diet was equivalent the relative feed intakes during the first and sixth to that of fish fed the reference diet. Feed intake by weeks of the experiment (Table 4). fish fed the Teo diet was significantly less than that of fish fed the reference diet. Feed intake by fish fed 3.2. Influence of gramine on feed digestibility diets that had a blend of Wodjil and Teo also had significantly poorer feed intake, but not as low as that Digestibility assessment of complete diets showed observed with Teo alone. that at low inclusion levels (b500 mg/kg), gramine

45 40 35 0 30 100 25 500 20 1000 15 10000 10

Feed Intake (g/tank/d) 5 0 1 2 3 4 5 6 7 8 9 Day post initial weighing

Fig. 1. Daily mean feed intake by tank, of each treatment, over the first 9 days of the experiment. Poorest feed intake was observed with the 10,000 mg/kg treatment, which was not significantly different from that of the 1500 mg/kg treatment. The 500 mg/kg treatment was significantly better than both the 1500 and 10,000 mg/kg treatments, but significantly poorer than the 100 mg/kg diet and the reference (0 mg/kg) treatments. No significant differences were noted between the reference and 100 mg/kg treatments. 518 B. Glencross et al. / Aquaculture 253 (2006) 512–522 did not influence the digestibility of nitrogen, energy growth levels observed of each treatment. No signifi- or phosphorus (Table 5). Because of poor diet palat- cant differences between the reference diet and all ability, sufficient faecal samples could not be obtained treatments up to and including 500 mg/kg were from the treatments with gramine levels higher than noted (Table 4). The FCR continued to increase with 500 mg/kg. increasing gramine level up to 1500 mg/kg. The Inclusion of the yellow lupin kernel meals (both 10,000 mg/kg treatment had negative growth and Wodjil and Teo varieties) into the diet did not sig- accordingly the fish had a negative FCR (Table 4). nificantly affect either the nitrogen or energy digest- The FCR of fish fed the Wodjil diet was not ibility, but significantly increased the digestibility of significantly different from that of the reference diet phosphorus in the diets compared to the reference diet (Table 4). However, the inclusion of Teo kernel meal (Table 5). resulted in a significantly poorer FCR and FCE. A blend of Teo and Wodjil resulted in FCR/FCE mid- 3.3. Influence of gramine on fish growth and feed way between that observed for the two discrete vari- utilisation eties (Table 4). Nitrogen and energy retention by fish fed the treat- Growth of fish fed the experiment treatments was ments was also largely consistent with feed intake. No largely consistent with feed intake. No effect on effect on nitrogen retention by the inclusion of gra- growth by the inclusion of gramine levels below mine below 1000 mg/kg levels was observed, how- 500 mg/kg levels was observed. From 500 mg/kg ever at 500 mg/kg a deterioration in the energy and above, a dramatic decline in growth was noted retention was noted relative to that of the reference (Table 4). This effect on growth was consistent for diet. From 1000 mg/kg and above, deterioration in both weight gain and DGC. A similar decline in both nitrogen and energy retention was noted (Table growth was noted with both of the negative controls 4). A similar decline in energy retention was noted (C1 and C2) (Table 4). Growth of fish fed the Wodjil with both of the negative controls (C1 and C2) (Table diet was not significantly different from that of the 4). Nitrogen and energy retention by fish fed the reference diet (Table 4). However, the inclusion of Wodjil diet was not significantly different from that Teo kernel meal significantly reduced growth. A blend of the reference diet (Table 4). However, the inclusion of Teo and Wodjil resulted in growth mid-way of Teo kernel meal significantly reduced retention between that observed for the two discrete varieties efficiency of both nitrogen and energy. A blend of (Table 4). Teo and Wodjil resulted in a significant reduction in Survival of fish was significantly reduced at gra- energy retention, but did not affect nitrogen retention mine inclusion levels above 1000 mg/kg. Poorer sur- (Table 4). vival was also noted from the Teo treatment (Table 4). The concentrations (pmol/l) of the thyroid hor- No other significant differences among treatments mones tri-iodothyronine (T3) and thyroxine (T4) were noted. of fish fed the treatments was also largely consistent 2 Feed conversion ratio (FCR) and feed conversion with feed intake (g/tank) ( yT3 =0.0565x +3.6343, R = 2 efficiency (FCE) were reflective of feed intake and 0.8441 and yT4 =0.0368x +1.6523, R =0.7634). No effect on either T3 or T4 concentrations was noted with the inclusion of gramine below 500 mg/kg levels. Table 5 From 500 mg/kg and above, deterioration in T con- Digestibility (%) of protein, energy and phosphorus from experi- 4 mental diets centrations were noted and above 1000 mg/kg a dete- Treatment ADC-protein ADC-energy ADC-phosphorus rioration in T3 concentrations was noted (Table 4). A similar decline in T3 and T4 concentrations was noted 0 87.2a 84.0a 29.0a 100 86.7a 86.0a 27.8a with the higher inclusion concentration of sulfamer- 500 88.0a 86.8a 31.6a azine sodium in the negative controls (C2), but not at a a b Wodjil 88.6 87.3 54.7 the lower inclusion concentration (C1) (Table 4). T4 Blend 87.2a 84.0a 51.6b concentrations from fish fed the Wodjil diet were Pooled S.E.M. 0.26 0.56 2.91 significantly less than those from fish fed the refer- B. Glencross et al. / Aquaculture 253 (2006) 512–522 519

ence diet, but no effects on T3 were noted (Table 4). Wallis test statistic =19.302) (Table 6). The difference However, the inclusion of Teo kernel meal signifi- is driven by treatments 6 and 7, where all readers cantly reduced both T3 and T4 concentrations. A blend awarded consistently high scores. of Teo and Wodjil resulted in a significant reduction in T4 concentrations, but did not affect T3 concentrations (Table 4). 4. Discussion

3.4. Influence of gramine on histology Any compound feed for an animal is generally only as valuable as the sum of the value of its ingre- The dark brown–black deposits did not stain for dients. The key value in an ingredient such as lupin iron or lipofuscin but did stain strongly for melanin. kernel meal is its protein and/or energy content. How- No lesions considered to represent significant changes ever, for most animals the use of plant protein in health status were detected in the liver, kidney, resources often introduces problems associated with spleen, pyloric caeca or intestine. Melano-macrophage the inherent anti-nutritional content of these resources. centres (MMC) are normally found in the kidney and Alkaloids have been touted as a potential anti-nutri- are characterised as dark brown–black macrophage tional problem with the use of lupin meals in aqua- aggregations of variable size and shape, however, culture diets, despite the previous lack of reliable data large variations were observed in the density of to confirm or refute this reputation (Francis et al., MMC in the haematopoetic tissue in the kidney sam- 2001). ples. These were scored independently and between reader scores were tested using Friedman two-way 4.1. Influence of gramine on feed intake ANOVA. There was no evidence of systematic variation between readers ( P b0.001, 2 df, Friedman test Alkaloids are generally believed to exert their anti- statistic =21.458). Variation in scores between treat- nutritional effect through inhibition of palatability at ments was significant for each reader (Reader 1, the lower inclusion concentrations, although other P b0.001, 11 df, Kruskal–Wallis test statistic=31.155; bioactive effects have been suggested at higher inclu- Reader 2, P b0.0001, 11 df, Kruskal–Wallis test sta- sion concentrations. The findings from the present tistic=38.826; Reader 3, P =0.056, 11 df, Kruskal– study confirm that above a critical threshold, the alkaloid gramine does have a strong anti-palatability Table 6 effect. The effect is noted at a minimum gramine Combined counts of scores (columns, 1=few, 4=abundant) concentration of 500 mg/kg of diet, though not at awarded by three independent readers to the number of melano- 100 mg/kg. A continuing strong anti-palatability macrophage centres in kidneys of fish in different treatments (rows, response is noted at higher inclusion concentrations 1–12) and at the maximum gramine inclusion concentration Scores examined in this study (10,000 mg/kg) insufficient Treatment 1 2 3 4 feed was consumed to even supply maintenance pro- 1 13 10 1 0 tein and energy demands. This compares well with 2 5 14 5 0 3 8 9 6 1 other species like rats, pigs and poultry (Pastuszewska 4 7 12 4 1 et al., 2001), but shows that fish are slightly more 5 4 7 8 5 sensitive in their palatability of gramine than either 6 0 4 15 5 pigs or poultry at least, and possibly rats too. 7 0 7 9 8 8 8 11 5 0 In undomesticated varieties of other lupin species, 9 10 11 2 1 such as L. angustifolius and L. cosentii, total alkaloid 10 8 10 3 0 concentrations exceeding 30,000 mg/kg have been 11 5 8 10 1 reported (Petterson, 2000). However, in Australia, 8 8 12 7 1 modern domesticated varieties of L. angustifolius Kidneys of eight fish were examined for each treatment except for are not made available for commercial release if treatment 10 (=7 fish). total alkaloid concentrations exceed 200 mg/kg (Glad- 520 B. Glencross et al. / Aquaculture 253 (2006) 512–522 stones, 1998; Perry et al., 1998). This has largely levels, with a certain degree of independence of the negated alkaloid related problems being observed in feed intake levels, suggests that there may be another animal feed industries, at least from Australian grown mechanism by which these meals are influencing the lupins. It should be noted that the L. angustifolius levels of this hormone. This contrasts results from (angustifoline, lupanine, a-isolupanine and 13- earlier work examining the use of L. luteus kernel hydroxy lupanine) and L. cosentii (epilupinine, epilu- meal, where no significant alterations to the thyroid pine-N-oxide and multiflorine) species of lupin have a hormones were noted (Glencross et al., 2004). How- totally different alkaloid profile to L. luteus. However, ever, in contrast to that study the present study used no fish feeding trials have been carried out using the plasma rather than whole blood samples and this may alkaloids in L. angustifolius species. have had significant effects on the reliability of the assays being used. The findings are also consistent 4.2. Influence of gramine on feed digestibility with work by Burel et al. (1998), who observed changes in thyroid hormone levels with the inclusion The observation that no effects on nitrogen, energy of L. albus kernel meal. Another study by Gomez et or phosphorus digestibility were seen at the 500 mg al. (1997), using commercial pellets showed no rela- gramine/kg diet inclusion concentration, relative to the tionship between plasma thyroid hormones and feed reference diet suggests that the alkaloid effect is not intake (%BW), though did show positive a relation- inhibiting the animal’s ability to absorb nutrients and ship against growth rate (SGR) in rainbow trout, energy from the diet once it is ingested. Although not similar to that observed in the present study. specifically related to the alkaloid effect, the inclusion of the yellow lupin kernel meals (both Wodjil and Teo 4.4. Influence of gramine on histology varieties) into the diet did improve the digestibility of phosphorus in the diets compared to the reference diet Melano-macrophage centres are normally found in and this has been noted in other studies on the digest- the liver and kidney of trout where they are involved ibility assessment of lupin kernel meals (Glencross and in trapping and removal of cellular debris and cellular Hawkins, 2004; Glencross et al., 2005). toxicants as well as storage of effete materials and recovery of iron (Agius, 1985; Agius and Roberts, 4.3. Influence of gramine on fish growth 2003). In this trial, there was an increase in the density of MMC with high dietary levels of gramine. MMC Growth, as assessed using a range of parameters increase in incidence with age, however, starvation, including weight gain, growth, nutrient and energy exposure to environmental contaminants and patholo- retention, of fish fed the experiment treatments was gical conditions resulting in cellular damage also largely consistent with feed intake. Survival of fish increase the incidence of MMC (Agius and Roberts, was also significantly reduced at gramine inclusion 2003; Wolke, 1992; Capps et al., 2004). The MMC in levels above 1000 mg/kg and was believed to result this trial were not associated with haemosiderin or from an inability of the fish to survive the experimen- lipofuscin but did stain strongly for melanin. Never- tal period with such a low level of feed intake. Feed theless, the density of MMC aggregations is a useful conversion ratio (FCR) and feed conversion efficiency bioindicator of fish health (Blazer et al., 1987; Capps (FCE) were also reflective of feed intake and growth et al., 2004). In the absence of any histological evi- levels observed of each treatment. dence for a toxic effect, it is likely that the increased That the levels of the plasma thyroid hormones tri- MMC densities observed in the fish fed high levels of iodothyronine (T3) and thyroxine (T4) of fish fed the gramine are associated with starvation. treatments were also largely consistent with feed intake across all experiment treatments suggests that the levels of these hormones are in response feed 5. Conclusion intake, not specifically the gramine levels. However, the observation that the inclusion of the L. luteus This study demonstrates that the lupin alkaloid kernel meals resulted in a significant change in T4 gramine can have a strong anti-nutritional effect on B. Glencross et al. / Aquaculture 253 (2006) 512–522 521 fish at certain critical inclusion levels. Although these Capps, T., Mukhi, S., Rinchard, J.J., Theodorakis, C.W., Blazer, V.S., inclusion levels exceed 100 mg/kg and are unlikely to Patin˘o, R., 2004. Exposure to perchlorate induces the formation of macrophage aggregates in the trunk kidney of zebrafish and be observed in diets even with 50% inclusion of mosquitofish. J. Aquat. Anim. Health 16, 145–151. kernel meals from Australian commercial varieties Carter, C.G., Hauler, R.C., 2000. Fish meal replacement by plant of either L. luteus or L. angustifolius. It is hypothe- meals in extruded feeds for Atlantic salmon, Salmo salar L. sised that the primary mode of action of gramine is Aquaculture 185, 299–311. through an anti-palatability effect that has secondary Fisher, D., 1996. Physiological variations in thyroid hormones: physiological and pathophysiological considerations. 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