Effect of a variety of animal, plant and single cell-based feed ingredients on diet digestibility and digestive enzyme activity in redclaw crayfish, Cherax quadricarinatus (Von Martens 1868)

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Aquaculture 272 (2007) 564 – 572 www.elsevier.com/locate/aqua-online

Effect of a variety of animal, plant and single cell-based feed ingredients on diet digestibility and digestive enzyme activity in redclaw crayfish, Cherax quadricarinatus (Von Martens 1868) Ana Pavasovic a , Alex J. Anderson b , Peter B. Mather a , Neil A. Richardson b,⁎ a

School of Natural Resource Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, Qld, Australia b School of Life Sciences, Queensland University of Technology, GPO Box2434, Brisbane, Qld, Australia Received 4 June 2007; received in revised form 7 August 2007; accepted 8 August 2007

Abstract Readily available agricultural products are often considered as feed ingredients when investigating cost-effective diet formulations for aquatic organisms. We investigated the potential use of fish meal (FM), meat and bone meal (MBM), poultry meal (PM), soybean meal (SBM), canola meal (CM), lupin meal (LM) and brewer's yeast (BY) in dietary formulations for redclaw crayfish, Cherax quadricarinatus. Test ingredients were incorporated at 30% inclusion level in a commercial redclaw pellet, and used in digestibility trials where apparent digestibility coefficients (ADCs) and digestive enzyme activities were measured and correlated. High ADC values were recorded for all treatments. The SBM diet had the highest apparent digestibility for dry matter (ADMD) (84.5%), crude protein (ACPD) (94.1%) and gross energy (AGED) (91.4%), while the lowest value was obtained for the diet containing MBM (75.9% ADMD; 88.5% ACPD; 85.5% AGED). Overall, ACPD was significantly higher (P b 0.05) for diets containing plant-based ingredients (93.5%) than for animal-based ones (90.0%). Similar trends were observed when dry matter and crude protein digestibilities were compared for specific feed ingredients. Specific activities of protease, amylase, cellulase and lipase in mid gut (MG) gland extracts were also determined for redclaw fed test diets. Generally, carbohydrase and lipase activities in individuals fed plant-based diets were significantly higher than in those fed animal-based diets. A significant correlation was observed for enzyme activity and ADC values. Protease activity was negatively correlated with diet ADMD, ingredient (I) ADMD, IACPD and IAGED, while lipase activity was positively correlated with ACPD and IACPD. Amylase/protease ratio (A/P) was positively correlated with all digestibility coefficients except AGED. Based on these observations, we conclude that redclaw have the capacity to successfully utilize nutrients from a broad range of dietary ingredients including animal, single cell and in particular, plant matter in their diet. We also suggest that digestive enzyme secretions and the ratio in which they occur, play a key role in determining digestibility of nutrients by redclaw. © 2007 Elsevier B.V. All rights reserved. Keywords: Cherax quadricarinatus; Redclaw; Digestive enzymes; Digestibility; Nutrition

1. Introduction ⁎ Corresponding author. Tel.: +61 7 3138 1388; fax: +61 7 3138 1534. E-mail address: [email protected] (N.A. Richardson). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.08.027

Redclaw, Cherax quadricarinatus, (Von Martens 1868) is a decapod crustacean (Decapoda; Parastacidae) endemic to freshwater river systems and lakes of

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northern Australia (Jones et al., 1998) and parts of Papua New Guinea (Holthuis, 1986). During the past 20 years, redclaw have been introduced to many countries and successful redclaw aquaculture industries established (Medley et al., 1994; Rouse, 1995; Romero, 1997; Chang, 2001). Suitability of redclaw for aquaculture can be attributed to a range of desirable traits (reviewed in García-Ulloa et al., 2003; Thompson et al., 2003; Campana-Torres et al., 2006) particularly a relatively simple life cycle (Morrissy, 1979) and an omnivorous feeding habit (Lawrence and Jones, 2002). Understanding the nutritional requirements of redclaw will be essential to ensuring profitable production and long-term sustainability of the redclaw aquaculture industry. Consequently, several studies have looked at specific aspects of redclaw nutrition, such as determining optimum dietary protein (Cortés-Jacinto et al., 2003, 2004; Thompson et al., 2004) and lipid levels (Hernandez et al., 2002; Hernandez-Vergara et al., 2003). Significant work has also been done to investigate the potential replacement of fish meal in artificial diets for redclaw (García-Ulloa et al., 2003; Muzinic et al., 2004; Thompson et al., 2005, 2006; Campana-Torres et al., 2005, 2006). This follows a global trend towards minimizing reliance on marine animal proteins (primarily fish meal) and use of cheaper alternatives. For many aquaculture species, fish meal replacement has become a priority issue due to increasing price of fish meal and unsustainable pressures on wild fisheries to satisfy demand for this product (Manzi, 1989; Hardy and Kissil, 1997; Naylor et al., 2000). Potentially viable alternatives to fish meal come from many agricultural commodities, particularly those with high protein content such as animal meals, oilseeds and grain legumes. Allan et al. (2000) identified the analysis of digestibility as the first step in estimating the potential of a new ingredient for use in artificial diets for aquaculture species. Studies that have investigated digestibility of various feed stuffs in freshwater crayfish are scarce however, despite the ability of crayfish to consume a wide range of food types. In general, natural food material consumed by freshwater crayfish consists of both animal and plant matter (Medley et al., 1994) with significant quantities of fungal and bacterial matter also ingested (Merrick, 1993). Campana-Torres et al. (2005) investigated protein and dry matter digestibility of some plant (soy paste, textured wheat, sorghum meal) and animal (sardine meal, squid meal and red crab meal) based ingredients in diets for juvenile redclaw. Their results show that redclaw appear to digest plant-based ingredients more efficiently than animal-based ones. This result was supported by Campana-Torres et al.

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(2006) who observed higher mean carbohydrate and lipid digestibility coefficients for plant-based ingredients than for animal-based ones. In contrast, Pavasovic et al. (2006) observed a significant reduction in digestibility of diets containing high levels of complex carbohydrates (cellulose). Nevertheless, very few studies have investigated digestibility coefficients for potential feed ingredients in redclaw. Hofer and Köck (1989) (c.f. Furne et al., 2005) observed that it is possible to predict the ability of a species to utilize different nutrients based on its digestive enzyme profile. Redclaw, like most other freshwater crayfish species possess a broad suite of digestive enzymes (Figueiredo et al., 2001). These have been investigated in a number of studies, that focused on the range of digestive enzymes secreted by redclaw (Figueiredo et al., 2001), ontogenetic changes in digestive protease and carbohydrase activities (Figueiredo and Anderson, 2003) and characterisation of cellulase activity (Xue et al., 1999). Attempts have also been made to investigate the effects of different nutrient sources on digestive enzyme activity in redclaw (Lopez-Lopez et al., 2005; Pavasovic et al., 2006). Findings from such studies are essential to help explain nutrient digestibility (Kolkovski, 2001) in aquatic organisms. In fish, data pertaining to digestive enzyme activity and profiles have helped overcome nutritional problems associated with formulation of artificial diets that best meet an animals' nutritive capability (Furne et al., 2005). They can also help define the inclusion limits for macronutrients such as dietary protein (Twining et al., 1983) and carbohydrates (Spannhof and Plantikow, 1983). Although digestive enzyme profiles may help to better explain nutrient use and relative digestibility, very few studies have investigated the ability of redclaw to utilize different nutrient sources by observing digestive enzyme responses. In this study, we analysed seven different potential feed ingredients that are available commercially by measuring digestibility coefficients and their relationship to digestive enzyme secretions in redclaw. 2. Materials and methods 2.1. Experimental animals and laboratory facility The adult redclaw (C. quadricarinatus), used in this study were obtained from a commercial crayfish producer, Sunshine Coast Crayfish Farmers (Yandina, Australia). Experiments were conducted in the recirculated aquaculture facility at the Queensland University of Technology, Brisbane, Australia. Prior to the commencement of the experiment, all experimental animals were weighed individually (average wet body weight 94.5 ± 3.5 g) and randomly assigned to treatments. Four crayfish were assigned per treatment diet (n = 4). The animals

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Table 1 List of ingredients and proximate composition of experimental diets Diet

Ingredient (%) Redclaw pellet Fish meal (FM) Meat and bone meal (MBM) Poultry meal (PM) Soybean meal (SBM) Canola meal (CM) Lupin meal (LM) Brewer's yeast (BY) Binder (wheat gluten) Common ingredients⁎ Proximate composition Crude protein (%) Crude ash (%) Gross energy (MJ/kg)

1

2

3

4

5

6

7

8

92.4

62.4 30

62.4

62.4

62.4

62.4

62.4

62.4

30 30 30 30 30 5 2.6

5 2.6

5 2.6

5 2.6

5 2.6

5 2.6

5 2.6

30 5 2.6

26.9 15.7 17.6

37.9 20.5 17.1

37.4 17.4 17.4

40.6 14.0 18.6

35.1 11.8 18.5

32.3 12.3 18.8

30.8 11.7 17.1

34.5 11.8 17.8

⁎Common ingredients: mineral and vitamin premix (2%), (kg − 1 of total diet − 4.68 g K2HPO4; 7.12 g MgSO4.7H2O; 1.84 g NaH2PO4.2H2O; vitamin premix (kg − 1)—100000 IU vitamin retinol; 500 mg thiamine; 1750 mg riboflavin; 1125 mg pyridoxine hydrochloride; 3750 mg cyanocobalamin; 25,000 mg ascorbic acid; 500 000 mg colecalciferol; 20000 IU d-alpha-tocopheryl acid succinate; 50 mg biotin); astaxanthin (0.1%); chromic oxide (Cr2O3) (0.5%).

were housed in individual glass tanks (160 ×160 × 220 mm). Optimum water quality conditions were maintained for the duration of the experiment with water temperature maintained at 27 ± 1 °C. All tanks were individually aerated to maintain dissolved oxygen concentrations above 4 mg/L and with photo-period set on a 12 h day/night cycle and start of the light phase at 0630 h each day. Faecal collection was commenced after the crayfish were fed experimental diets for 7 days. 2.2. Diets and digestibility determinations A digestibility trial was conducted to evaluate the digestibility coefficients of test ingredients: South American fish meal (FM) (Ridley Aqua-Feed, Australia), meat and bone meal (MBM) (Southern Meats, Australia), poultry meal (PM) (AJ Bush, Australia), soybean meal (SBM) (Radford Park Aquafeed, Australia), canola meal (CM) (Radford Park Aquafeed, Australia), lupin meal (LM) (MC Croker, Australia) and brewer's yeast (BY) (Swift and Co., Australia). A reference diet was used as a control and it consisted of a commercial redclaw pellet (Radford Park Redclaw Grower Pellet, Australia), with the following proximate nutrient composition: 89.4% dry matter, 20% crude protein, 3.4% crude lipid and 4.2% crude fibre. Experimental diets were formulated by combining test ingredients and the reference diet in a 30:70% ratio, on a dry weight basis. A complete list of ingredients for all diets, including their proximate composition is presented in Table 1. Table 2 contains information on the proximate composition of test ingredients used in experimental diets. Diets used in the experiment were prepared by thoroughly mixing dry ingredients, followed by wet ingredients, until a crumbly dough

consistency was achieved. Diet mixture was pressure pelleted using a meat grinder with a 3 mm die. Pellets were steamed in a microwave oven (Sharp) for 2.5 min prior to drying at 50 °C in a drying oven, overnight. All experimental diets were stored at −20 °C until required. The proximate composition of diets and energy and protein content of faecal material were determined at the Animal Research Institute (Brisbane, Australia) according to AOAC protocols (1984). Chromic oxide (Cr2O3) was added to all diets as an inert marker used to estimate digestibility coefficients. Cr content of diets and faecal material used in calculating apparent digestibility values were determined using a method described by Furukawa and Tsukahara (1966).

Table 2 Proximate composition of the test ingredients used in the digestibility trial on C. quadricarinatus Ingredient Dry Crude matter (%) protein (%)

Crude Crude Gross energy fat (%) ash (%) (MJ/kg)

FM MBM PM SBM CM LM BY

8.7 13.4 13.1 1.9 3.8 9.4 0.4

91.7 97.7 96.7 88.3 90 86.1 95.1

75.5 59.6 69.2 53.2 44.1 30.8 48.6

17.2 20.4 14.1 7.2 7.4 3.6 9.6

15.95 16.93 20.9 20.59 21.6 15.93 18.26

FM = fish meal, MBM = meat and bone meal, PM = poultry meal, SBM = soybean meal, CM = canola meal, LM = lupin meal, BY = brewer's yeast.

A. Pavasovic et al. / Aquaculture 272 (2007) 564–572 Table 3 Apparent digestibility coefficients for dry matter (ADMD), crude protein (ACPD) and gross energy (AGED) of the test diets for C. quadricarinatus Diet

Apparent digestibility coefficients (%) ADMD

ACPD

AGED

Reference FM MBM PM SBM CM LM BY Average value for all animalbased diets Average value for all plantbased diets

82.08 ± 0.35ab 81.39 ± 1.05ab 75.89 ± 2.36c 80.48 ± 2.36ab 84.51 ± 0.14a 79.33 ± 1.45bc 82.68 ± 0.68ab 83.27 ± 1.61ab 79.26 ± 1.27a

93.58 ± 0.44a 91.48 ± 0.45bc 88.48 ± 0.85d 90.13 ± 0.99cd 94.08 ± 0.24a 92.44 ± 0.34ab 93.95 ± 0.20a 93.14 ± 0.63ab 90.03 ± 0.56b

90.16 ± 1.18ab 89.22 ± 0.65abc 85.48 ± 1.22d 88.34 ± 1.11bc 91.40 ± 0.23a 86.92 ± 1.07cd 90.00 ± 0.42ab 89.04 ± 0.63abc 87.68 ± 0.72a

82.17 ± 2.89a

93.49 ± 0.26a

89.44 + 0.67a

Values represent mean ± standard error (n = 4). FM = fish meal, MBM = meat and bone meal, PM = poultry meal, SBM = soybean meal, CM = canola meal, LM = lupin meal, BY = brewer's yeast. Means displaying different superscripts within the same column are significantly different (P b 0.05).

Apparent dry matter (ADMD), crude protein (ACPD) and gross energy (AGED) digestibilities were calculated using the following equations, as suggested by Jones and De Silva (1998): ADMD = 100–100 (%marker in feed/%marker in faeces); ACPD = 100–100 [(%marker in feed/%marker in faeces) × (%crude protein in faeces/%crude protein in feed)]. AGED = 100–100 [(%marker in feed/%marker in faeces) × (gross energy in faeces/gross energy in feed)]. Apparent digestibility coefficients of the test ingredients were calculated using the following equations described by Bureau et al. (1999). ADCI = ADCT + ((1−s) DR/s DI) (ADCT−ADCR); where: ADCI = apparent digestibility coefficient of test ingredient; ADCT = apparent digestibility coefficient of test diet; ADCR = apparent digestibility coefficient of the reference diet; DR = % nutrient (or kJ/g gross energy) of the reference diet mash; DI = % nutrient (or kJ/g gross energy) of the test ingredient; s = proportion of test ingredient in test diet mash (i.e. 0.3 in this study); (1−s) = proportion of reference diet mash in test diet mash (i.e. 0.7 in this study). 2.3. Enzymatic determinations All animals used in the digestibility trial were assayed for protease, amylase, cellulase and lipase activity. Upon completion of the digestibility study, all the test animals were euthanized 180 min after final feeding by immersion in ice water. The hepatopancreas (or midgut gland (MG)) was then immediately removed from each animal and homogenised according to the methods described by Pavasovic et al. (2006). Homogenates were stored frozen at −20 °C until required. Assays for total protein, protease, amylase and cellulase activity were performed according to methods described by Pavasovic et al.

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(2006). Lipase activity was analysed according to the method described by Vega-Villasante et al. (1999). Briefly, the assay was performed by first preparing the mixture consisting of 100 μL of sodium taurocholate (200 mM), 1.9 mL of TrisHCl (50 mM, pH 7.5) and 20 μL of the hepatopancreatic extract (1/20 dilution). This mixture was incubated for 5 min at 37 °C then 20 μL of octanoate substrate (200 mM in dimethyl sulfoxide-DMSO) was added and the assay mixture incubated for a further 30 min at 37 °C. Next, 20 μL of Fast Blue BB solution (100 mM in DMSO) was added to the mixture and incubated for 5 min at 37 °C. Reactions were stopped by adding 200 μL of TCA (0.72 M) and the solution clarified with 2.71 mL of ethyl acetate/ethanol (1:1). Absorbance of the solution was measured at 540 nm using a spectrophotometer (Novaspec II, Pharmacia LKB Technology, Uppsala, Sweden). For all assays, one enzyme unit (U) was defined as the quantity of enzyme that catalysed the release of 1 μmol of product per minute under the assay conditions. In this study specific enzyme activity was defined as enzyme units (U) per mg of protein. 2.4. Statistical analyses Data from the different treatment groups were analysed using one-way ANOVA, set at the P b 0.05 significance level. Where significant differences were observed, least-significant difference (LSD) a post hoc test was applied for multiple comparisons. Prior to analysis of variance, data were tested for equality of variance using a homogeneity of variance test. Spearman's rank correlation coefficient was used to identify significant correlations between ADC coefficients and specific digestive enzyme activity. All statistical analyses were carried out using SPSS (v.15). 3. Results 3.1. Digestibility determinations Apparent digestibility coefficients for dry matter (ADMD), crude protein (ACPD) and gross energy (AGED) obtained for test diets are presented in Table 3. ADMD coefficients ranged from 75.9% to 84.5%. The highest ADMD value was obtained with the SBM diet which was significantly higher than those obtained for diets containing either CM or MBM. By contrast, the lowest ADMD value was obtained with the MBM diet which was significantly lower (Pb 0.05) than all other diets tested except the diet containing CM. Apparent digestibility of protein was relatively high for all treatments and ranged 88.5% to 94.1%. Significantly, ACPD coefficients for diets containing plant-based test ingredients were all higher (Pb 0.05) than diets containing terrestrial animal-based meals. AGED values for test diets ranged from 85.5% to 91.4%. The highest AGED value was obtained using SBM diet that was significantly higher (PN 0.05) than values obtained using diets with terrestrial animal-based meals or CM. Apparent dry matter and crude protein digestibility coefficients calculated for specific feed ingredients are presented in Table 4. The highest ADMD value was obtained for SBM and this was significantly higher than the value obtained for MBM

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Table 4 Apparent digestibility coefficients for dry matter (ADMD), crude protein (ACPD) and gross energy (AGED) of the test ingredients for C. quadricarinatus Ingredient

FM MBM PM SBM CM LM BY Average value for all animal ingredients Average value for all plant ingredients

Apparent digestibility coefficients (%) ADMD

ACPD

AGED

80.17 ± 3.04ab 65.32 ± 6.20c 77.87 ± 6.50ab 89.29 ± 0.41a 74.45 ± 4.93bc 83.83 ± 0.20ab 85.62 ± 2.37ab 74.46 ± 3.47a

89.80 ± 0.80bc 83.43 ± 1.69d 87.16 ± 1.85c 94.61 ± 0.51a 90.95 ± 0.79b 94.60 ± 0.56a 92.61 ± 1.38ab 86.80 ± 1.12b

86.87 ± 2.28abcd 74.47 ± 4.11e 84.88 ± 3.22cd 93.81 ± 0.69a 80.93 ± 3.04de 89.63 ± 1.46abc 86.61 ± 2.01abcd 82.08 ± 2.37a

82.52 ± 2.38a

93.39 ± 0.62a

88.13 ± 1.92a

Values represent mean ± standard error (n = 4). FM = fish meal, MBM = meat and bone meal, PM = poultry meal, SBM = soybean meal, CM = canola meal, LM = lupin meal, BY = brewer's yeast. Values displaying different superscripts within the same column are significantly different (P b 0.05).

or CM. The highest ACPD value was also obtained for SBM which was significantly greater than for the CM or both terrestrial animal meal diets. Interestingly, the average ACPD value obtained for plant-based ingredients was significantly higher than that obtained for animal-based ingredients. The ingredient with the highest AGED value was SBM (93.81%) while the lowest value was recoded for MBM (74.5%) which was significantly lower than all other ingredients, except CM (80.9%). 3.2. Enzymatic determinations Specific enzyme activities for protease, amylase, cellulase and lipase across all treatments are presented in Table 5.

Highest protease activity (5.6 × 10− 3 U/mg protein) was observed in individuals fed the diet with CM. Moreover this value was significantly higher than protease activity levels in redclaw fed diets containing SBM (2.3 × 10− 3 U/mg protein), LM (2.4 × 10− 3 U/mg protein) or BY (2.5 × 10− 3 U/mg protein). A significant amylase activity was detected across all treatments. Overall, the amylase activity levels were approximately one thousand fold higher than those detected for protease. The highest level of amylase activity was detected in individuals given the CM diet (5.6 U/mg protein), which, was significantly higher than that for redclaw fed the MBM diet (2.8 U/mg protein). The diet containing CM also displayed the highest level of cellulase activity which was significantly above the values obtained using diets based on terrestrial animal meals. Moreover, for all treatments, cellulase activity levels in mid gut gland extracts were approximately ten fold higher than amylase activities. Lipase activity was highest in redclaw fed diets containing SBM (7.1 U/mg protein) or LM (6.7 U/mg protein). Lipase values obtained using SBM and LM diets were significantly higher (P b 0.05) that that obtained in redclaw fed the MBM diet (3.4 U/mg protein). Overall, digestive enzyme activity, including the A/P ratio, was significantly higher in animals fed diets high in plantbased ingredients than animal-based ones, for all enzymes except protease. ADC values were correlated with digestive enzyme activities to determine if a relationship existed between diet digestibility and redclaw digestive secretions (Table 6). Spearman rank correlation revealed a very strong positive correlation between amylase and protease activity, while cellulase activity was also strongly correlated with both protease and amylase activity. Lipase activity however, was not significantly correlated with any other enzyme activities. A significant correlation was observed for enzyme activity and apparent digestibility coefficients. Protease was negatively correlated with ADMD, IADMD, IACPD and IAGED. Lipase was positively correlated with ACPD and IACPD, while amylase/protease ratio (A/P)

Table 5 Digestive enzyme activity in MG extracts from C. quadricarinatus fed different experimental diets Diet

Reference FM MBM PM SBM CM LM BY Animal-based diets Plant-based diets

Enzyme Protease⁎

Amylase

Cellulase

Lipase

A/P#

4.34 ± 0.93 3.64 ± 0.91 3.60 ± 0.54 3.96 ± 0.52 2.34 ± 0.30b 5.61 ± 1.04a 2.44 ± 0.54b 2.53 ± 0.48b 3.74 ± 0.36a 3.46 ± 0.58a

4.36 ± 0.77 3.63 ± 0.45 2.76 ± 0.56b 3.54 ± 0.74 4.48 ± 1.11 5.64 ± 1.19a 4.87 ± 1.29 3.85 ± 0.36 3.31 ± 0.33b 5.00 ± 0.61a

40.59 ± 7.1 34.07 ± 4.3 28.07 ± 4.9bc 25.87 ± 4.6c 43.04 ± 3.8ab 45.78 ± 6.3a 37.89 ± 7.1 32.73 ± 3.7 29.34 ± 2.6b 42.24 ± 3.2a

6.19 ± 1.57 4.61 ± 0.32 3.39 ± 0.48b 4.24 ± 1.60 7.10 ± 0.56a 6.53 ± 0.81 6.72 ± 1.21a 5.05 ± 1.43 4.08 ± 0.54b 6.78 ± 0.48a

1.12 ± 0.29cd 1.15 ± 0.25cd 0.78 ± 0.15d 0.87 ± 0.08d 1.89 ± 0.38a 0.99 ± 0.06cd 2.05 ± 0.34a 1.62 ± 0.19abc 0.93 ± 0.10b 1.64 ± 0.21a

⁎ (×10− 3); # (×103). Values presented are mean ± standard error (n = 4) for specific activity of protease, amylase, cellulase and lipase, expressed as U/mg protein. FM = fish meal, MBM = meat and bone meal, PM = poultry meal, SBM = soybean meal, CM = canola meal, LM = lupin meal, BY = brewer's yeast, A/P = amylase/ protease ratio. Means displaying different superscripts within the same column are significantly different (P b 0.05).

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Table 6 Correlation matrix of specific enzyme activity and apparent digestibility coefficients for C. quadricarinatus Protease r Protease Amylase Cellulase Lipase ADMD IADMD ACPD IACPD AGED IAGED

Amylase P

1

− 0.398⁎ − 0.426⁎ − 0.324 − 0.467⁎ −0.338 − 0.504⁎⁎

r 0.399⁎ 1

0.024 0.024 0.070 0.012 0.058 0.006

0.029 0.101 0.184 0.169 − 0.016 0.024

Cellulase

Lipase

A/P

P

r

P

r

P

r

P

0.024

0.475⁎⁎ 0.636⁎⁎ 1

0.006 0.000

0.216 0.363 0.207

0.873 0.610 0.312 0.391 0.929 0.903

0.023 0.019 0.289 0.230 0.154 0.094

0.902 0.925 0.108 0.239 0.401 0.636

− 0.225 0.166 0.229 1 0.157 0.204 0.365⁎ 0.437⁎ 0.198 0.325

− 0.589⁎⁎ 0.455⁎⁎ 0.152 0.398⁎ 0.438⁎ 0.513⁎⁎ 0.547⁎⁎ 0.667⁎⁎ 0.313 0.504⁎⁎

0.000 0.009 0.407 0.024 0.012 0.005 0.001 0.000 0.081 0.006

0.392 0.299 0.040 0.020 0.278 0.091

⁎Correlation is significant at the 0.05 level. ⁎⁎Correlation is significant at the 0.01 level. ADMD = apparent dry matter digestibility of diet; IADMD = apparent dry matter digestibility of ingredient; ACPD = apparent crude protein digestibility; IACPD = apparent crude protein digestibility of ingredient; AGED = apparent gross energy digestibility of diet; IAGED = apparent gross energy digestibility of ingredient; A/P = amylase/protease ratio.

was strongly correlated with ADMD, IADMD, ACPD, IACPD and IAGED.

4. Discussion In the present study, we observed high ADC values for all test ingredients and corresponding diets, reflecting the ability of adult redclaw to utilize a wide range of nutrient sources. High digestibility coefficients for a wide variety of animal and plant-based ingredients are not unusual in freshwater crayfish, as observed in juvenile redclaw (Campana-Torres et al., 2005), red swamp crayfish (Procambarus clarkii) (Reigh et al., 1990) and yabby (Cherax destructor) (Jones and De Silva, 1997). The finding that plant-based ingredients and brewer's yeast were effectively digested by redclaw in the present study is in agreement with the findings of CampanaTorres et al. (2005) who observed high ADC values in juvenile redclaw fed plant-based ingredients such as soy paste. High digestibility of SBM in the current study is also consistent with similar reports for the closely related C. destructor (Jones and De Silva, 1997). In P. clarkii, however, all ADC values for SBM, except for protein digestibility of individual ingredient, were marginally lower (77% ADMD, 78% IADMD, 92% ACPD, 82% ADE) than was observed for redclaw here (Reigh et al., 1990). Other crustaceans, including Litopenaeus vannamei (Amaya et al., 2007), Penaeus setiferus (Brunson et al., 1997) and Macrobrachium rosenbergii (Law et al., 1990), also have been shown to effectively utilize soy products in their diet. Relatively high protein and energy digestibility coefficients for diets containing SBM and LM in the present study, indicate that redclaw have the ability to successfully

utilize these plant-based ingredients. By contrast, relatively low ADC values were obtained using diets containing CM. Elsewhere, it has been suggested that high levels of lignin (∼ 11.3%) in canola meal lower its digestibility (Hertrampf and Piedad-Pascual, 2000). This may account for the significantly lower dry matter and crude protein digestibility of CM relative to SBM observed here. Sudaryono et al. (1996) observed low ADCs (53.2% ADMD and 85.4% APD) for diets containing lupin seed meal given to P. monodon, but later reported higher digestibilities (71% ADMD and 89% APD) (Sudaryono et al., 1999), with the authors attributing the differences in digestibility to differences in the lupin species and meal processing. Muzinic et al. (2004) report that brewer's grains with yeast can be given to redclaw at dietary inclusion levels of up to 30%, without adverse effects on growth and survival. This agrees with high digestibility for BY observed here. By contrast, Reigh et al. (1990) reported relatively low ADC values for yeast (61.6–72.1% for dry matter and 69.8–84.5% for protein) in P. clarkii. The high digestibility of BY observed in the current investigation, suggests that there is potential to utilize yeast products successfully in redclaw diets, and should therefore be considered as a potential feed supplement. In the present study, two terrestrial animal-based ingredients (MBM and PM) demonstrated consistently lower nutrient digestibilities than plant or yeast based ingredients. Although, this is in general agreement with results achieved by Campana-Torres et al. (2005), comparisons are difficult because different protein sources and inclusion levels of the test ingredients were used. ADC values for MBM and the MBM diet here are consistent with those observed for P. clarkii (Reigh

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et al., 1990). Reduced digestibility coefficients have been associated with use of MBM or meat meal in C. destructor (Jones and De Silva, 1997) and P. setiferus (Brunson et al., 1997). Reduced digestibility related to the use of MBM is often attributed to high levels of ash (Wu et al., 1999; Allan et al., 2000) and may explain very low digestibility coefficients for MBM observed in redclaw here. Despite this, MBM has been used in crustacean diets where it was used successfully to replace 60% (Tan et al., 2005) to 75% (Forster et al., 2003) of FM in the diet for L. vannamei, and up to 50% of FM protein in diets for Macrobrachium nipponense (Yang et al., 2004) without a significant negative effect on growth and survival. Use of PM in the present study, although it did not significantly affect ADMD, resulted in significantly reduced APD, particularly relative to plant and yeast ingredients. Reduced protein digestibility has often been attributed to contamination of the PM with raw feathers (Hertrampf and Piedad-Pascual, 2000). The range of digestive enzyme activity observed in the present study agrees in general with data reported in other studies (Figueiredo et al., 2001; Lopez-Lopez et al., 2005; Pavasovic et al., 2006). Redclaw appear to have the ability to modify their digestive enzyme secretions in response to different ingredients in the diet over time (Lopez-Lopez et al., 2005). Interestingly, when treatment diets were grouped based on ingredient type (i.e. plant vs animal) activity of all non-protease enzymes was significantly higher in redclaw fed diets containing plant-based ingredients. By contrast, a lack of a clear correlation between plant and animal feed ingredients and protease activity was reported by LopezLopez et al. (2005), who recorded highest protease activity in redclaw fed a sardine meal diet and soy paste diet. Likewise, no significant difference was observed in protease activity under any dietary treatments for Scylla serrata (Pavasovic et al., 2004) and L. vannamei (RivasVega et al., 2006). Protease activity, however, may be influenced by nutrient quality and quantity (Rodríguez et al., 1994; Le Moullac et al., 1996). Lemos et al. (2000) observed diverse characteristics of digestive proteinases in terms of molecular weight, in four species of marine prawns, suggesting that protease activity may differ significantly among species. High levels of secretion of carbohydrate degrading enzymes, such as amylase and cellulase, observed for redclaw here are typical of omnivorous and herbivorous organisms that are generally considered to utilize higher levels of carbohydrates than carnivorous animals (Hidalgo et al., 1999; Furne et al., 2005). Our results show that amylase and cellulase activity is significantly higher in

animals fed diets containing plant-based ingredients which typically contain a higher proportion of carbohydrates. Interestingly, Lopez-Lopez et al. (2005) found no correlation between amylase activity and carbohydrate content in the diet of redclaw and attributed this to the use of different sources of starch. Digestive enzyme activities revealed that both amylase and cellulase activities were inversely correlated with protease activity in redclaw. Guzman et al. (2001) also reported a strong correlation between amylase and protease activity in L. setiferus, however the opposite was reported by Gamboa-Delgado et al. (2003), who observed no significant correlation between amylase and protease activity in L. vannamei. Lipase activity was not correlated significantly with any other enzyme type, although a strong positive relationship was observed with A/P ratio. In fact, A/P ratio was significantly correlated with all enzyme groups measured here, except cellulase. The ratio of amylase to protease secretion probably reflects the feeding patterns of individual crustacean species, with enzyme activity related to substrates most common in the diet (Figueiredo and Anderson, 2003). In crustaceans, a change in A/P ratio has generally been associated with ontogeny, where individuals consume less protein in latter stages of life (GamboaDelgado et al., 2003). In the present study, the A/P ratio was significantly higher in animals fed high levels of plant-based ingredients and was strongly correlated with diet digestibility coefficients. These findings reflect the omnivorous feeding habit of redclaw, and provide evidence that the A/P ratio significantly affects nutrient utilization in adult crayfish. In the current study strong correlations were also demonstrated between the activity levels of individual enzymes and digestibility coefficients. For example, for all test ingredients protease activity was inversely related to ADMD, IADMD, IACPD and IAGED. Lipase activity also strongly correlated with apparent protein digestibility of both, diets and ingredients. Little is understood about how lipase contributes to digestibility in crustaceans. For example, Lopez-Lopez et al. (2005) observed significant differences in lipase activity in test animals fed a sorghum diet and those containing red crab meal and sardine meal, but failed to explain the reason for such variation. Based on the findings of our study, redclaw can successfully digest a range of commercially available ingredients, in particular those of plant origin. Our results demonstrate a strong relationship between the digestive enzymes secreted by redclaw and nutrient digestibility which needs to be further investigated to allow development of cost-effective, nutritious artificial feeds, specifically formulated for this species.

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Acknowledgments The authors would like to thank Mr. Marko Pavasovic and Dr. Peter J. Prentis for their technical assistance during the experiments. We are also grateful to Ross and Vicki Rickard of Sunshine Coast Crayfish Farmers (Yandina, Australia) for their cooperation and supply of redclaw crayfish during this project. Our gratitude is also extended to Dr Richard Smullen of Ridley Aqua-Feed (Australia) for supplying the fishmeal and Mr John Harsant of Radford Park Aquafeeds (Australia) for supplying the soybean, canola and cotton seed meals. This manuscript is written in partial fulfillment of the Doctor of Philosophy degree in aquaculture of Ana Pavasovic, at the Queensland University of Technology, Brisbane, Australia. References Allan, G.L., Parkinson, S., Booth, M.A., Stone, D.A.J., Rowland, S.J., Frances, J., Warner-Smith, R., 2000. Replacement of fish meal in diets for Australian silver perch, Bidyanus bidyanus: I. Digestibility of alternative ingredients. Aquaculture 186, 293–310. Amaya, E.A., Davis, D.A., Rouse, D.B., 2007. Replacement of fish meal in practical diets for the Pacific white shrimp (Litopenaeus vannamei) reared under pond conditions. Aquaculture 262, 393–401. AOAC, 1984. Official methods of analysis, 14th edn. Association of Official Analytical Chemists, Washington, DC, USA. Brunson, J.F., Romaire, R.P., Reigh, R.C., 1997. Apparent digestibility of selected ingredients in diets for white shrimp Penaeus setiferus L. Aquac. Nutr. 3, 9–16. Bureau, D.P., Harris, A.M., Cho, C.Y., 1999. Apparent digestibility of rendered animal protein ingredients for rainbow trout (Oncorhynchus mykiss). Aquaculture 180, 345–358. Campana-Torres, A., Martinez-Cordova, L.R., Villarreal-Colmenares, H., Civera-Cerecedo, R., 2005. In vivo dry matter and protein digestibility of three-plant derived and four animal-derived feedstuffs and diets for juvenile Australian redclaw, Cherax quadricarinatus. Aquaculture 250, 748–754. Campana-Torres, A., Martinez-Cordova, L.R., Villarreal-Colmenares, H., Civera-Cerecedo, R., 2006. Carbohydrate and lipid digestibility of animal and vegetal ingredients and diets for juvenile Australian redclaw crayfish, Cherax quadricarinatus. Aquac. Nutr. 12, 103–109. Chang, A.K.W., 2001. Analysis of the performance of a formulated feed in comparison with a commercial prawn feed for the crayfish, Cherax quadricarinatus. World Aquac. 32, 19–23. Cortés-Jacinto, E., Villarreal-Colmenares, H., Civera-Cerecedo, R., Martínez-Córdova, L., 2003. Effect of dietary protein level on growth and survival of juvenile freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae). Aquac. Nutr. 9, 207–213. Cortés-Jacinto, E., Villarreal-Colmenares, H., Civera-Cerecedo, R., Naranjo-Páramo, J., 2004. Effect of dietary protein level on the growth and survival of pre-adult freshwater crayfish Cherax quadricarinatus (von Martens) in monosex culture. Aquac. Res. 35, 71–79. Figueiredo, M.S.R.B., Anderson, A.J., 2003. Ontogenetic changes in digestive proteases and carbohydrases from the Australian

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