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Effect of dietary protein level on spawning and egg quality of redclaw crayfish Cherax quadricarinatus
Aquaculture 257 (2006) 412 – 419 www.elsevier.com/locate/aqua-online
Effect of dietary protein level on spawning and egg quality of redclaw crayfish Cherax quadricarinatus Hervey Rodríguez-González a , Manuel García-Ulloa b , Alfredo Hernández-Llamas a , Humberto Villarreal a,⁎ a
Programa de Acuacultura, Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo Santa Rita, La Paz, B.C.S. 23090, México b Laboratorio de Ciencias Marinas, Universidad Autónoma de Guadalajara. López de Legazpi 235, Apdo. Postal 3, Barra de Navidad, Jalisco 48987, México Received 19 July 2005; received in revised form 18 January 2006; accepted 18 January 2006
Abstract The effect of dietary protein level on spawning and egg quality was evaluated for female Cherax quadricarinatus. Diets containing four different levels of crude protein were evaluated (22, 27, 32, and 37%). After 100 days, no significant effects of protein level were found on survival (78.6–84.5%), final weight (41.0–43.1g), or fecundity (8.5–9.2 eggs/g female). The percentage of spawning females ranged from 19.7 to 27.3%, and a significant fit, using a quadratic equation estimated maximum spawning to occur at 30% crude protein. Significantly greater egg area (3.90 mm2), volume (39.3 mm3), weight (5.44 μg), and diameter (2.27 mm) were observed at 32% crude protein. There were no significant differences in mean egg protein (2227.1 ± 445.0 μg/egg), lipid (430.9 ± 85.2 μg/egg) and carbohydrate (73.9 ± 10.6 μg/egg) contents, and energy (13.3 ± 2.1 kcal/egg) in relation to dietary protein level. High statistical power indicated that biochemical composition was not affected by dietary protein level. We conclude that a dietary crude protein content of 32% is recommended for reproduction of female redclaw crayfish. © 2006 Elsevier B.V. All rights reserved. Keywords: Redclaw crayfish; Cherax quadricarinatus; Dietary protein; Spawning; Egg quality
1. Introduction Aquaculture production of Australian redclaw crayfish is expanding in Australia (Jones, 1995), Ecuador (Romero, 1997), Thailand and Malaysia (Chang, 2001), United States (Masser and Rouse, 1993), and Mexico (Villarreal and Peláez, 1999).
⁎ Corresponding author. Tel.: +52 612 123 8484; fax: +52 612 125 3625. E-mail address: [email protected] (H. Villarreal). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.01.020
Several authors (e.g. Mills and McCloud, 1983; Villarreal and Peláez, 1999; Jones, 1995) have demonstrated that production of Cherax spp. can be increased when formulated feeds are used. Reports on the use of experimental diets for C. quadricarinatus (Meade and Watts, 1995; Cortés-Jacinto et al., 2003, 2005; Hernandez-Vergara et al., 2003) outline basic nutritional requirements for growout. However, very little information on requirements for optimum egg quality production of broodstock exists. Several studies have evaluated maturation, eye ablation (Sagi et al., 1997), use of hormones (Abdu et
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al., 2001), fecundity (King, 1993), and reproductive cycle (Villarreal et al., 1999; Serrano-Pinto et al., 2004) of redclaw under laboratory conditions. Different diets have been used in these studies. Diet plays an important role in crayfish broodstock condition (Holdich, 2002). Broodstock nutrition is important for reproductive success because egg and larval production are strongly dependent on the diets offered (Bromage, 1995; Harrison, 1997; García-Ulloa, 2000). Protein is the most critical ingredient in practical diets because it is expensive and growth responses are affected (Cortés-Jacinto et al., 2003; Thompson et al., 2005). According to Harrison (1997), protein required in broodstock diets for maturation and production of eggs is higher than the level required for growout because gonad maturation is a process of intense protein synthesis, mainly during vitellogenesis (Abdu et al., 2000). An understanding of protein requirements for spawning and subsequent progeny production is necessary to improve culture practices. However, no investigations addressing this concern have been conducted with redclaw. This study evaluated the effect of different
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dietary protein levels on broodstock spawning and egg quality. 2. Materials and methods 2.1. Diet preparation Four diets were formulated by using Mixit-Win software (Agricultural Software Consultants Inc., San Diego, CA, USA). Crude protein levels in diets were 22, 27, 32, and 37% (Table 1). The ingredients were ground and passed through a 0.5-mm mesh sieve and then mixed thoroughly. Each diet was prepared by mixing all the macro-ingredients in an industrial blender (Thunderbird®, Blaine, WA, USA). The micro-ingredients were pre-mixed manually in a plastic container before being added. Fish oil and soy lecithin were homogenized until an emulsion was obtained and then added to the mixture. Water was added to make a stiff-like dough that was passed through a meat grinder (Tor-Rey™, Monterrey, N. L., México) to pelletize the mixture. The resulting 2-mm pellets were dried in a forced-air oven (Hafo Series 1600, VWR 1680) at 40 °C for 8 h. Pellets
Table 1 Ingredient composition (g/kg) and proximate analysis (%dry weight) of the experimental diets for female broodstock Cherax quadricarinatus Ingredient a
Sardine meal Sorghum meala Soybean meala Squid meala Wheat meala Fish oila Soy lecithina Binder (grenetine) a Mineral premixb Vitamin premixc Ascorbic acidd Choline chloridee Calcium carbonatef Proteinsg Ether extract g Moistureg Ashg Crude Fiberg N-free extract Gross energy (kJ/g)g a
22% CP
27% CP
32% CP
37% CP
45.8 429.2 150.0 30.0 200.0 32.1 32.1 40.0 25.0 3.5 1.5 0.6 10.0 22.62 ± 0.14 7.37 ± 0.2 9.91 ± 0.27 6.51 ± 0.02 – 63.5 20.45 ± 0.02
129.2 345.8 150.0 30.0 200.0 32.1 32.1 40.0 25.0 3.5 1.5 0.6 10.0 27.65 ± 0.08 8.03 ± 0.03 8.47 ± 0.19 7.33 ± 0.01 0.96 ± 0.03 56.03 20.19 ± 0.03
213.3 261.8 150.0 30.0 200.0 32.1 32.1 40.0 25.0 3.5 1.5 0.6 10.0 32.38 ± 0.29 7.73 ± 0.03 8.78 ± 0.11 8.71 ± 0.22 0.28 ± 0.01 50.89 20.74 ± 0.02
298.0 177.1 150.0 30.0 200.0 32.1 32.1 40.0 25.0 3.5 1.5 0.6 10.0 37.18 ± 0.09 8.21 ± 0.02 8.01 ± 0.05 10.00 ± 0.03 0.42 ± 0.01 44.19 21.61 ± 0.02
Source: PIASA®, La Paz, B.C.S., México. Mineral mix (g kg− 1 diet): KCl, 0.5; MgSO4.7H2O, 0.5; ZnSO4.7H2O, 0.09; MnCl2.4H2O, 0.00234; CuSO4.5H2O, 0.005; KI, 0.005; CoCl2·2H2O, 0.00025; Na2HPO4, 2.37. c Vitamin mix (units in mg/kg except where given): retinol, 5000 IU; cholecalciferol, 4000 IU; α-tocopherol acetate, 100; menadione, 5; thiamin, 60; riboflavin, 25; pyridoxine HCl, 50; pantothenic acid, 75; niacin, 40; biotin, 1; inositol, 400; cyanocobalamin, 0.2; folic acid, 10. d Stay C (35% active agent) Roche®. e Choline chloride (65% active agent). f Acs reagent. SIGMA®. g Mean ± SD. b
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were then packed in plastic bags and refrigerated at 5 °C until used. Proximate composition of diets was determined according to AOAC (1995). Crude protein was determined using the micro-Kjeldahl method (APHA, 1989) and crude lipids were extracted with anhydrous ether in a soxhlet extraction system (Soxtec Avanti 2050, Foss Tecator®, Copenhague, Denmark) following Bligh and Dyer (1959). Ash was determined by using a muffled furnace (AOAC, 1995), and crude fiber by the phenol-sulfuric acid method (Myklestad and Haug, 1972). Gross energy of the diets was determined using an adiabatic bomb calorimeter (Model 1261, Parr, Moline, IL, USA). 2.2. Feeding trial Pre-adult redclaw crayfish, Cherax quadricarinatus (23.0 ± 3.0 g), were acclimated to laboratory conditions in twelve 1500-l fiberglass tanks (125 pre-adults per tank) for 1 week. During this time crayfish were fed with a commercial diet for shrimp (35% CP, PIASA®, La Paz, Mexico). After acclimation, animals were weighed and sexed according to Villarreal and Peláez (1999), and labeled with permanent ink. Females were stocked at a density of 9.7 animals/m2 (223 g/m2) into 1500-l fiberglass tanks (two replicates per treatment were randomly allocated) and feeding with experimental diet for 45 days. After this period, males were stocked to establish a relation of 3 females to 1 male. The system was static and a 30% water exchange was done daily at 10 am. Temperature was maintained at 28 ± 1 °C using 300-W heaters (Aquarium Pharmaceuticals, Inc., Paris, France). A 5-hp blower (Sweetwater®, Apopka, FL, USA) and air diffusers maintained dissolved oxygen above 5 mg/l; a 14:10-h day/night photoperiod was used. To provide refuge, 2 bundles composed of five sheets of open weave synthetic mesh and 25 PVC pipes (50 mm diameter, 200 mm in length) were placed in each tank. 2.3. Data collection Experimental tanks (8) were observed daily to find spawning females. Twelve berried females per dietary treatment were selected during the feeding trial to collect eggs and evaluate their biochemical composition. Selected females were weighed using a digital balance (0.01 g, Navigator, Oahus®, Florham Park, New Jersey, USA), and measured (± 0.01 mm), and each specimen placed in individual 80-l fiberglass aquaria. Twenty eggs were carefully detached from the pleopods of each female by using dissecting clasps. The sampled eggs
were used for studying individual wet and dry weights, minor and major diameter, area, volume, and biochemical composition. To determine fecundity (eggs/g female), all the eggs were removed from the female and counted. Final percent survival of females was calculated. For biochemical analyses, eggs were homogenized in a saline solution (1.2% NaCl). To quantify protein, each homogenate was first digested with 0.5 N NaOH. Protein concentration was determined by the Bradford Method (1976) using albumin as standard, and absorbance was read at 595 nm, using a Spectron Genesys Spectrophotometer. To determine the level of carbohydrate, proteins were precipitated with 20% trichloroacetic acid and centrifuged at 2608×g for 10 min at 4 °C. Carbohydrates were then quantified from a sample of the supernatant by the Anthrone Method (Van Handel, 1965), using glucose as standard. Absorbance was read at 620 nm. For lipids, an adaptation of the method of Barnes and Blackstock (1973) was used. An aliquot of the homogenate was mixed with analytical grade H2SO4 and incubated at 80 °C for 10 min. The acid solution obtained was mixed with the sulphosphophovanillin reagent and absorbance was recorded using a Biorad 560 microplate reader at 560 nm, using a mixture of triacylglycerols (12 mg/ml) and cholesterol (8 mg/ml) as standard (Palacios et al., 2000). To calculate the values for gross energy, the conversion factors suggested by Heras et al. (1998) were applied (17.9 J/mg for protein, 38.4 J/mg for lipid, and 17.1 J/mg for carbohydrate). 2.4. Statistical analysis Normality and variance homogeneity of data were evaluated with Lilliefor's and Bartlett's Tests (Sokal and Rohlf, 2000). Percentages of survival and spawning were transformed to arcsine values prior to analysis. Regression analysis was used to determine the response spawning females (percentage) to dietary protein level. A quadratic equation was used for regression (Shearer, 2000), as follows. PE ¼ a0 þ a1 S þ a2 S 2
ð1Þ
where a0, a1, a2 are regression coefficients and S is the percent of substitution. The percent of substitution yielding the optimum percentage of spawning (PSm) was calculated from the previous equations as: PSm ¼ a1 ð2a2 Þ1
ð2Þ
One-way ANOVA was used to evaluate the effect of protein level on the remainder of the variables studied.
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Diet (% CP)
Weight (g)
Survival (%)
Fecundity (egg/g female)
22 27 32 37
41.54 ± 0.1 41.42 ± 1.5 41.08 ± 1.7 43.11 ± 5.2
84.5 ± 1.6 78.6 ± 13.4 82.1 ± 1.6 84.5 ± 1.6
9.12 ± 2.01 8.50 ± 0.14 9.28 ± 0.66 9.10 ± 3.18
No significant differences were found (P > 0.05).
ANOVA was followed by the post-hoc Duncan's Multiple Test to separate significantly different mean values. When a lack of significance for biochemical composition of eggs was found using ANOVA, power analysis was conducted to determine the probability of not accepting a false null hypothesis (Cohen, 1998; Searcy-Bernal, 1994). Significance level was set at P = 0.05 and power at 0.95 (i.e. β = 0.05). Effect size for power analysis was estimated from results obtained by Rodríguez-González (2001) for significant differences in the contents of protein, lipids, carbohydrates, and energy in the gonad, in response to protein dietary content. Procedures available in STATISTICA 6® and G*Power 2 (StatSoft®) were used to conduct the analyses. 3. Results No significant differences were found in survival (78.6–84.5%), final weight (41.0–43.1g), and fecundity (8.5–9.2 eggs/g female) of female redclaw crayfish using the different levels of dietary protein (Table 2). A significant regression analysis showed a satisfactory fit of the quadratic equation to percentage of spawning females. The protein level yielding the maximum percentage was 30% (Fig. 1). The percentage of spawning females ranged from 19.7 to 27.3%. There were significant differences in morphological features of eggs. Egg area ranged from 3.60 to 3.90 mm2, volume from 34.5 to 39.3 mm3, weight from 4.68 to 5.44 mg, and diameter from 2.17 to 2.27 mm. Maximum values were consistently present in crayfish fed the 32% protein diet (Fig. 2). Energy, mean protein, lipid, or carbohydrate contents did not differ significantly relative to dietary protein level. High statistical power (> 0.95) indicated that protein, lipid, and carbohydrate levels in the egg were unaffected by the dietary level of protein (Table 3).
4. Discussion Dietary protein influenced the proportion of redclaw crayfish spawners and the size and weight of their eggs. The role of dietary nutrients for broodstock, such as protein, has been studied for other crustaceans (Tacon and Cowey, 1985; Watanabe et al., 1985). Gonad maturation, fecundity, and quality of both eggs and juveniles of aquatic organisms are influenced by broodstock nutrition (Bromage, 1995; Rodríguez-González, 2001; Wouters et al., 2001). Protein is a structural, functional, and energy constituent of tissues, and plays an important role in spawning, fertilization, and normal development of the embryo in decapods (Harrison, 1990; Wouters et al., 2001; García-Guerrero et al., 2003). Several authors (i.e. Yeh and Rouse, 1994; Barki et al., 1997; Abdu et al., 2001; McPhee et al., 2004) have presented spawning rates for the species. The values reported in the present study are within the range reported, and comparable to those indicated by Yeh and Rouse (1995), for similar density, sex ratio and water temperature. This study shows that a dietary protein level of about 30% maximizes the size of the spawning population. This suggests that such protein level is optimal to meet nutritional requirements for reproductive females. While there are no previous studies determining optimal dietary protein requirement for spawning female redclaw crayfish, these are similar to recommendations made for growout of pre-adults (27%
35 30 25 Spawning (%)
Table 2 Mean final weight, survival and fecundity of female Cherax quadricarinatus fed diets containing different dietary levels of protein (22, 27, 32, and 37% CP) for 75 days
415
20 15 10 2
s=-0.12CP +7.17CP-80.97
5
2
R = 0.96 0 17
22
27
32
37
42
% Crude Protein
Fig. 1. Mean spawning (s) percentage of Cherax quadricarinatus females fed with four crude protein (CP) levels (22, 27, 32, and 37%) for 75 days. The crude protein yielding the maximum percentage of spawning was calculated from regression coefficients as: 7.17/ (2 × 0.12) = 30 (dashed line).
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A
B 2.45
6.0
2.00
2.75
a
a 2.70
2.30 2.25
4.5 a
2.20
ab
4.0
b
Wet weight Mean diameter
b
22
27
32
37
b
b
1.90
2.60 2.55
1.85
a
2.50 2.45 2.40
2.10
2.35
1.80
Major diameter
1.75
b
b
2.15
ab
Minor diameter
3.5 17
Major diameter (mm)
Mean diameter (mm)
Wet weight (mg)
2.35
b b
5.0
1.95
ab 2.65
Minor diameter (mm)
2.40 ab
5.5
1.70 17
42
22
27
% Crude Protein
32
37
42
% Crude Protein
C 46
4.1
a
4.0
44
3.9
ab
2
Area (mm )
3
b
b
40
3.7 3.6
38
a
3.5
Volume (mm )
42 3.8
36
ab
3.4 3.3
Volume
b
3.2 17
22
34
Area
b
32 27
32
37
42
% Crude Protein
Fig. 2. Mean wet weight, diameter (A), major and minor diameter (B), area and volume (C) of eggs from female Cherax quadricarinatus fed diets containing different dietary protein levels (22, 27, 32, and 37%).
crude protein; Cortés-Jacinto et al., 2004) and juveniles (32% crude protein; Cortés-Jacinto et al., 2005). The growth rates in our trial were smaller than those reported by Cortés-Jacinto et al. (2003), Thompson et al. (2003),
and Thompson et al. (2005) for juveniles. However, they were comparable to those reported by Cortés-Jacinto et al. (2004), for similar sized organisms. The different protein levels used in this experiment yielded a
Table 3 Mean biochemical composition (n = 12) of eggs from Cherax quadricarinatus females fed diets containing different levels of dietary protein (22, 27, 32, and 37%) Diet (%CP)
Protein (μg/egg)
Lipid (μg/egg)
Carbohydrate (μg/egg)
Energy (J/egg)
22 27 32 37 Statistical power
2107.70 ± 544.3 2369.730 ± 320.3 2176.660 ± 530.4 2238.510 ± 331.3 0.96
440.260 ± 80.1 408.430 ± 93.3 422.150 ± 74.6 463.080 ± 94.1 0.99
70.690 ± 6.9 69.230 ± 10.7 78.600 ± 10.1 75.530 ± 12.2 0.98
53.700 ± 4.3 57.760 ± 7.4 55.590 ± 9.2 55.220 ± 6.4 0.99
No significant differences were found (P > 0.05).
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fecundity value (8.5–9.2 eggs/g) similar to that reported by Yeh and Rouse (1994) (7.8–10 egg/g female). Egg quality is defined as the ability of an egg to be fertilized and undergo development (Holcomb et al., 2004). Morphological features of eggs, hatching rates, biochemical composition, and levels of specific nutrients are generally deemed to be good indicators of egg quality (Clarke et al., 1990; Lavens et al., 1991; Watanabe et al., 1991; García-Ulloa et al., 2004). In our study, mean egg diameter, volume, area, and weight were higher for the 32% crude protein diet. This value closely approximates the one calculated to maximize the size of spawning population, suggesting that 32% crude protein in the diet is optimal for obtaining high quality eggs. Information on morphological features of C. quadricarinatus eggs is scarce, however, the mean newly laid egg size obtained in our study (2.60 mm) coincides with the size of oocytes before laying (2.55 mm) reported by Sagi et al. (1996). We found that protein, lipid, carbohydrate, and energy in eggs were not affected by dietary protein level and that, for the conditions studied, biochemical composition cannot be used as an indicator of egg quality. Two factors may be responsible for this response. García-Guerrero et al. (2003) stated that the biochemical composition of eggs in C. quadricarinatus is dependent on the nutritional status of the female. The reproductive strategy of the species also plays a role. The redclaw crayfish has relatively low fecundity and exhibits maternal care (Holdich, 2002). Under this strategy, it is hypothesized the reproductive effort of the female is aimed at guaranteeing uniform egg quality and reproductive success by using different nutrient sources, and that spawning occurs only after a balanced and uniform biochemical composition of eggs is obtained. From the results in the present study, it can be concluded that a dietary protein content of 32% is recommended for reproduction of female redclaw C. quadricarinatus. However the actual value may vary according to dietary ingredient composition, digestibility (Campaña-Torres et al., 2005) and methodology used for protein determination. This information should contribute to the development of adequately balanced practical diets for female redclaw crayfish broodstock. Acknowledgements The authors thank Beatriz Romero, Francisco Encarnación, and Ismael Valdivia for assistance during the trials, and Sonia Rocha and Dolores Rondero for the biochemical analyses. The editor at CIBNOR provided
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assistance with improving the English text. This study was supported by CONACYT grant U39531-Z and CIBNOR grant AC.2.1 to Humberto Villarreal. Hervey Rodríguez is a recipient of a CONACYT doctoral fellowship. References Abdu, U., Yehezkel, G., Sagi, A., 2000. Oocyte development and polypeptide dynamics during ovarian maturation in the red claw crayfish Cherax Quadricarinatus. Invertebr. Reprod. Dev. 37, 75–83. Abdu, U., Barki, A., Karplus, I., Barel, S., Takac, P., Yehezkel, G., Laufer, H., Sagi, A., 2001. Physiological effects of methyl farnesoate and pyriproxyfen on wintering female crayfish Cherax quadricarinatus. Aquaculture 202, 163–175. AOAC, 1995. Official Methods of Analysis of the Association of Analytical Chemist, 16th ed. AOAC International, Washington, DC, USA. 935 pp. APHA, 1989. Standard Methods for the Examination of Water and Wastewater, 14th ed. American Public Health Association, Washington, DC, USA. Barki, A., Levi, T., Hulata, G., Karplus, I., 1997. Annual cycle of spawning and molting in the red claw crayfish, Cherax quadricarinatus, under laboratory conditions. Aquaculture 157, 239–249. Barnes, H., Blackstock, J., 1973. Estimation of lipid in marine animals and tissues: detailed investigation of sulphophosphovanillin method for total lipids. J. Exp. Mar. Biol. Ecol. 12, 103–118. Bligh, E.G., Dyer, W.I., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Bradford, M., 1976. A rapid sensitive method for the quantification of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72, 248–254. Bromage, N., 1995. Broodstock management and seed quality— general considerations. In: Bromage, N.R., Roberts, R.J. (Eds.), Broodstock Management and Egg and Larval Quality. Blackwell Science, Oxford, UK, pp. 1–24. Campaña-Torres, A., Martinez-Cordova, L., 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. 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. Clarke, A., Brown, J.H., Holmes, L.J., 1990. The biochemical composition of eggs from Macrobrachium rosenbergii in relation to embryonic development. Comp. Biochem. Physiol. 96B, 505–511. Cohen, J., 1998. Statistical Power Analysis for the Behavioral Sciences. Lawrence Erlbaum Associates Publishers, Hillsdale, NJ, USA. 567 pp. Cortés-Jacinto, E., Villarreal-Colmenares, H., Civera-Cerecedo, R., Martínez-Cordova, R., 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
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Mills, B.J., McCloud, P.I., 1983. Effects of stocking and feeding rates on experimental pond production of the crayfish Cherax destructor Clark (Decapoda: Parastacidae). Aquaculture 34, 51–72. Myklestad, S., Haug, A., 1972. Production carbohydrates by the marine diatom Chaetoceros affini var. wille (Gran) Husted: I. Effect of the concentration of nutrients in the culture medium. J. Expl. Mar. Biol. Ecol. 9, 125–136. Palacios, E., Ibarra, A.M., Racotta, I.S., 2000. Tissue biochemical composition in relation to multiple spawning in wild and pondreared Penaeus vannamei broodstock. Aquaculture 185, 353–371. Rodríguez-González, H., 2001. Effect of protein and lipids content in the gonad development of females of the freshwater Australian crayfish Cherax quadricarinatus (Von Martens). Master's Thesis, Centro de Investigaciones Biológicas del Noroeste, La Paz, B.C.S., Mexico. 86 pp. Romero, X.M., 1997. Production of redclaw crayfish in Ecuador. World Aquac. 28, 5–10. Sagi, A., Shokrum, R., Isam, K., Rise, M., 1996. Gonad maturation, morphological and physiological changes during the first reproductive cycle of the crayfish Cherax quadricarinatus female. Invertebr. Reprod. Dev. 29, 235–242. Sagi, A., Shoukrun, R., Levy, T., Barki, A., Hulata, G., Karplus, I., 1997. Reproduction and molt in previously spawned and first-time spawning red claw crayfish Cherax quadricarinatus females following eyestalk ablation during the winter reproductive-arrest period. Aquaculture 156, 101–111. Searcy-Bernal, R., 1994. Statistical power and aquaculture research. Aquaculture 127, 371–388. Serrano-Pinto, V., Landais, I., Ogliastro, M.H., Gutierrez-Ayala, M., Mejia-Ruiz, H., Villarreal-Colmenares, H., García-Gasca, A., Vazquez-Boucard, C., 2004. Vitellogenin mRNA expression in Cherax quadricarinatus during secondary vitellogenic at first maturation females. Mol. Reprod. Dev. 69, 17–21. Shearer, K.D., 2000. Experimental design, statistical analysis and modelling of dietary nutrient requirement studies for fish: a critical review. Aquac. Nutr. 6, 91–102. Sokal, R.R., Rohlf, F.J., 2000. Biometry, The Principles and Practice of Statistics in Biological Research, 3rd. ed. Freeman and Company, New York, USA. 887 pp. Tacon, A.G., Cowey, C.B., 1985. Protein and amino acid requirements. Aquacult. Dev. Coord. Programme. FAO, Rome, Italy. Thompson, K.R., Muzinic, L.A., Cristian, T.D., Webster, C.D., Manomaitis, L., Rouse, D.B., 2003. Effect on growth, survival, and fatty acid composition of Australian red claw crayfish Cherax quadricarinatus, fed practical diets with and without supplemental lecithin and/or cholesterol. J. World Aquac. Soc. 34, 1–10. Thompson, K.R., Muzinic, L.A., Engler, L.S., Webster, C.D., 2005. Evaluation of practical diets containing different protein levels, with or without fish meal, for juvenile Australian red claw crayfish (Cherax quadricarinatus). Aquaculture 244, 241–249. Van Handel, E., 1965. Estimation of glycogen in small amounts of tissue. Anal. Biochem. 11, 256–265. Villarreal, H., Naranjo, J., Hinojosa, P., 1999. Effect of density on the growth, survival and biomass production of the Australian freshwater crayfish Cherax quadricarinatus (redclaw) in gravellined ponds in Ecuador. Abstract p. 795, World Aquaculture 1999, The Annual International Conference and Exposition of the World Aquaculture Society. Sydney, Australia. Villarreal, H., Peláez, J., 1999. Biología y Cultivo de Langosta de Agua Dulce Cherax quadricarinatus. Centro de Investigaciones Biológicas del Noroeste and Acuacultivos Santo Domingo, La Paz, B.C.S., México.
H. Rodríguez-González et al. / Aquaculture 257 (2006) 412–419 Watanabe, T., Koizumi, T., Suzuki, H., Satoh, S., Takeuchi, T., Yoshida, N., Kitada, T., Tsukashima, Y., 1985. Improvement of quality of red sea bream eggs by feeding broodstock on a diet containing cuttlefish meal on raw krill shortly before spawning. Bull. Jpn. Soc. Sci. Fish. 51, 1511–1521. Watanabe, T., Lee, M., Mizutani, J., Yamada, T., Satoh, S., Takeuchi, T., Yoshida, N., Kitada, T., Arakawa, T., 1991. Effective components in cuttlefish meal and raw krill for improvement of quality of red seabream Pagrus major eggs. Nippon Suisan Gakkaishi 57, 681–694. Wouters, R., Molina, C., Lavens, P., Calderón, J., 2001. Lipid composition and vitamin content of wild female Litopenaeus
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vannamei in different stage of sexual maturation. Aquaculture 198, 307–323. Yeh, H.S., Rouse, D.B., 1994. Indoor spawning and egg development of the red claw crayfish Cherax quadricarinatus. J. World Aquacult. Soc. 25, 297–301. Yeh, H.S., Rouse, D.B., 1995. Effects of water temperature, density, and sex ratio on the spawning rate of red claw crayfish Cherax quadricarinatus (von Martens). J. World Aquacult. Soc. 26, 160–164.
Effect of dietary protein level on spawning and egg quality of redclaw crayfish Cherax quadricarinatus Hervey Rodríguez-González a , Manuel García-Ulloa b , Alfredo Hernández-Llamas a , Humberto Villarreal a,⁎ a
Programa de Acuacultura, Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo Santa Rita, La Paz, B.C.S. 23090, México b Laboratorio de Ciencias Marinas, Universidad Autónoma de Guadalajara. López de Legazpi 235, Apdo. Postal 3, Barra de Navidad, Jalisco 48987, México Received 19 July 2005; received in revised form 18 January 2006; accepted 18 January 2006
Abstract The effect of dietary protein level on spawning and egg quality was evaluated for female Cherax quadricarinatus. Diets containing four different levels of crude protein were evaluated (22, 27, 32, and 37%). After 100 days, no significant effects of protein level were found on survival (78.6–84.5%), final weight (41.0–43.1g), or fecundity (8.5–9.2 eggs/g female). The percentage of spawning females ranged from 19.7 to 27.3%, and a significant fit, using a quadratic equation estimated maximum spawning to occur at 30% crude protein. Significantly greater egg area (3.90 mm2), volume (39.3 mm3), weight (5.44 μg), and diameter (2.27 mm) were observed at 32% crude protein. There were no significant differences in mean egg protein (2227.1 ± 445.0 μg/egg), lipid (430.9 ± 85.2 μg/egg) and carbohydrate (73.9 ± 10.6 μg/egg) contents, and energy (13.3 ± 2.1 kcal/egg) in relation to dietary protein level. High statistical power indicated that biochemical composition was not affected by dietary protein level. We conclude that a dietary crude protein content of 32% is recommended for reproduction of female redclaw crayfish. © 2006 Elsevier B.V. All rights reserved. Keywords: Redclaw crayfish; Cherax quadricarinatus; Dietary protein; Spawning; Egg quality
1. Introduction Aquaculture production of Australian redclaw crayfish is expanding in Australia (Jones, 1995), Ecuador (Romero, 1997), Thailand and Malaysia (Chang, 2001), United States (Masser and Rouse, 1993), and Mexico (Villarreal and Peláez, 1999).
⁎ Corresponding author. Tel.: +52 612 123 8484; fax: +52 612 125 3625. E-mail address: [email protected] (H. Villarreal). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.01.020
Several authors (e.g. Mills and McCloud, 1983; Villarreal and Peláez, 1999; Jones, 1995) have demonstrated that production of Cherax spp. can be increased when formulated feeds are used. Reports on the use of experimental diets for C. quadricarinatus (Meade and Watts, 1995; Cortés-Jacinto et al., 2003, 2005; Hernandez-Vergara et al., 2003) outline basic nutritional requirements for growout. However, very little information on requirements for optimum egg quality production of broodstock exists. Several studies have evaluated maturation, eye ablation (Sagi et al., 1997), use of hormones (Abdu et
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al., 2001), fecundity (King, 1993), and reproductive cycle (Villarreal et al., 1999; Serrano-Pinto et al., 2004) of redclaw under laboratory conditions. Different diets have been used in these studies. Diet plays an important role in crayfish broodstock condition (Holdich, 2002). Broodstock nutrition is important for reproductive success because egg and larval production are strongly dependent on the diets offered (Bromage, 1995; Harrison, 1997; García-Ulloa, 2000). Protein is the most critical ingredient in practical diets because it is expensive and growth responses are affected (Cortés-Jacinto et al., 2003; Thompson et al., 2005). According to Harrison (1997), protein required in broodstock diets for maturation and production of eggs is higher than the level required for growout because gonad maturation is a process of intense protein synthesis, mainly during vitellogenesis (Abdu et al., 2000). An understanding of protein requirements for spawning and subsequent progeny production is necessary to improve culture practices. However, no investigations addressing this concern have been conducted with redclaw. This study evaluated the effect of different
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dietary protein levels on broodstock spawning and egg quality. 2. Materials and methods 2.1. Diet preparation Four diets were formulated by using Mixit-Win software (Agricultural Software Consultants Inc., San Diego, CA, USA). Crude protein levels in diets were 22, 27, 32, and 37% (Table 1). The ingredients were ground and passed through a 0.5-mm mesh sieve and then mixed thoroughly. Each diet was prepared by mixing all the macro-ingredients in an industrial blender (Thunderbird®, Blaine, WA, USA). The micro-ingredients were pre-mixed manually in a plastic container before being added. Fish oil and soy lecithin were homogenized until an emulsion was obtained and then added to the mixture. Water was added to make a stiff-like dough that was passed through a meat grinder (Tor-Rey™, Monterrey, N. L., México) to pelletize the mixture. The resulting 2-mm pellets were dried in a forced-air oven (Hafo Series 1600, VWR 1680) at 40 °C for 8 h. Pellets
Table 1 Ingredient composition (g/kg) and proximate analysis (%dry weight) of the experimental diets for female broodstock Cherax quadricarinatus Ingredient a
Sardine meal Sorghum meala Soybean meala Squid meala Wheat meala Fish oila Soy lecithina Binder (grenetine) a Mineral premixb Vitamin premixc Ascorbic acidd Choline chloridee Calcium carbonatef Proteinsg Ether extract g Moistureg Ashg Crude Fiberg N-free extract Gross energy (kJ/g)g a
22% CP
27% CP
32% CP
37% CP
45.8 429.2 150.0 30.0 200.0 32.1 32.1 40.0 25.0 3.5 1.5 0.6 10.0 22.62 ± 0.14 7.37 ± 0.2 9.91 ± 0.27 6.51 ± 0.02 – 63.5 20.45 ± 0.02
129.2 345.8 150.0 30.0 200.0 32.1 32.1 40.0 25.0 3.5 1.5 0.6 10.0 27.65 ± 0.08 8.03 ± 0.03 8.47 ± 0.19 7.33 ± 0.01 0.96 ± 0.03 56.03 20.19 ± 0.03
213.3 261.8 150.0 30.0 200.0 32.1 32.1 40.0 25.0 3.5 1.5 0.6 10.0 32.38 ± 0.29 7.73 ± 0.03 8.78 ± 0.11 8.71 ± 0.22 0.28 ± 0.01 50.89 20.74 ± 0.02
298.0 177.1 150.0 30.0 200.0 32.1 32.1 40.0 25.0 3.5 1.5 0.6 10.0 37.18 ± 0.09 8.21 ± 0.02 8.01 ± 0.05 10.00 ± 0.03 0.42 ± 0.01 44.19 21.61 ± 0.02
Source: PIASA®, La Paz, B.C.S., México. Mineral mix (g kg− 1 diet): KCl, 0.5; MgSO4.7H2O, 0.5; ZnSO4.7H2O, 0.09; MnCl2.4H2O, 0.00234; CuSO4.5H2O, 0.005; KI, 0.005; CoCl2·2H2O, 0.00025; Na2HPO4, 2.37. c Vitamin mix (units in mg/kg except where given): retinol, 5000 IU; cholecalciferol, 4000 IU; α-tocopherol acetate, 100; menadione, 5; thiamin, 60; riboflavin, 25; pyridoxine HCl, 50; pantothenic acid, 75; niacin, 40; biotin, 1; inositol, 400; cyanocobalamin, 0.2; folic acid, 10. d Stay C (35% active agent) Roche®. e Choline chloride (65% active agent). f Acs reagent. SIGMA®. g Mean ± SD. b
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were then packed in plastic bags and refrigerated at 5 °C until used. Proximate composition of diets was determined according to AOAC (1995). Crude protein was determined using the micro-Kjeldahl method (APHA, 1989) and crude lipids were extracted with anhydrous ether in a soxhlet extraction system (Soxtec Avanti 2050, Foss Tecator®, Copenhague, Denmark) following Bligh and Dyer (1959). Ash was determined by using a muffled furnace (AOAC, 1995), and crude fiber by the phenol-sulfuric acid method (Myklestad and Haug, 1972). Gross energy of the diets was determined using an adiabatic bomb calorimeter (Model 1261, Parr, Moline, IL, USA). 2.2. Feeding trial Pre-adult redclaw crayfish, Cherax quadricarinatus (23.0 ± 3.0 g), were acclimated to laboratory conditions in twelve 1500-l fiberglass tanks (125 pre-adults per tank) for 1 week. During this time crayfish were fed with a commercial diet for shrimp (35% CP, PIASA®, La Paz, Mexico). After acclimation, animals were weighed and sexed according to Villarreal and Peláez (1999), and labeled with permanent ink. Females were stocked at a density of 9.7 animals/m2 (223 g/m2) into 1500-l fiberglass tanks (two replicates per treatment were randomly allocated) and feeding with experimental diet for 45 days. After this period, males were stocked to establish a relation of 3 females to 1 male. The system was static and a 30% water exchange was done daily at 10 am. Temperature was maintained at 28 ± 1 °C using 300-W heaters (Aquarium Pharmaceuticals, Inc., Paris, France). A 5-hp blower (Sweetwater®, Apopka, FL, USA) and air diffusers maintained dissolved oxygen above 5 mg/l; a 14:10-h day/night photoperiod was used. To provide refuge, 2 bundles composed of five sheets of open weave synthetic mesh and 25 PVC pipes (50 mm diameter, 200 mm in length) were placed in each tank. 2.3. Data collection Experimental tanks (8) were observed daily to find spawning females. Twelve berried females per dietary treatment were selected during the feeding trial to collect eggs and evaluate their biochemical composition. Selected females were weighed using a digital balance (0.01 g, Navigator, Oahus®, Florham Park, New Jersey, USA), and measured (± 0.01 mm), and each specimen placed in individual 80-l fiberglass aquaria. Twenty eggs were carefully detached from the pleopods of each female by using dissecting clasps. The sampled eggs
were used for studying individual wet and dry weights, minor and major diameter, area, volume, and biochemical composition. To determine fecundity (eggs/g female), all the eggs were removed from the female and counted. Final percent survival of females was calculated. For biochemical analyses, eggs were homogenized in a saline solution (1.2% NaCl). To quantify protein, each homogenate was first digested with 0.5 N NaOH. Protein concentration was determined by the Bradford Method (1976) using albumin as standard, and absorbance was read at 595 nm, using a Spectron Genesys Spectrophotometer. To determine the level of carbohydrate, proteins were precipitated with 20% trichloroacetic acid and centrifuged at 2608×g for 10 min at 4 °C. Carbohydrates were then quantified from a sample of the supernatant by the Anthrone Method (Van Handel, 1965), using glucose as standard. Absorbance was read at 620 nm. For lipids, an adaptation of the method of Barnes and Blackstock (1973) was used. An aliquot of the homogenate was mixed with analytical grade H2SO4 and incubated at 80 °C for 10 min. The acid solution obtained was mixed with the sulphosphophovanillin reagent and absorbance was recorded using a Biorad 560 microplate reader at 560 nm, using a mixture of triacylglycerols (12 mg/ml) and cholesterol (8 mg/ml) as standard (Palacios et al., 2000). To calculate the values for gross energy, the conversion factors suggested by Heras et al. (1998) were applied (17.9 J/mg for protein, 38.4 J/mg for lipid, and 17.1 J/mg for carbohydrate). 2.4. Statistical analysis Normality and variance homogeneity of data were evaluated with Lilliefor's and Bartlett's Tests (Sokal and Rohlf, 2000). Percentages of survival and spawning were transformed to arcsine values prior to analysis. Regression analysis was used to determine the response spawning females (percentage) to dietary protein level. A quadratic equation was used for regression (Shearer, 2000), as follows. PE ¼ a0 þ a1 S þ a2 S 2
ð1Þ
where a0, a1, a2 are regression coefficients and S is the percent of substitution. The percent of substitution yielding the optimum percentage of spawning (PSm) was calculated from the previous equations as: PSm ¼ a1 ð2a2 Þ1
ð2Þ
One-way ANOVA was used to evaluate the effect of protein level on the remainder of the variables studied.
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Diet (% CP)
Weight (g)
Survival (%)
Fecundity (egg/g female)
22 27 32 37
41.54 ± 0.1 41.42 ± 1.5 41.08 ± 1.7 43.11 ± 5.2
84.5 ± 1.6 78.6 ± 13.4 82.1 ± 1.6 84.5 ± 1.6
9.12 ± 2.01 8.50 ± 0.14 9.28 ± 0.66 9.10 ± 3.18
No significant differences were found (P > 0.05).
ANOVA was followed by the post-hoc Duncan's Multiple Test to separate significantly different mean values. When a lack of significance for biochemical composition of eggs was found using ANOVA, power analysis was conducted to determine the probability of not accepting a false null hypothesis (Cohen, 1998; Searcy-Bernal, 1994). Significance level was set at P = 0.05 and power at 0.95 (i.e. β = 0.05). Effect size for power analysis was estimated from results obtained by Rodríguez-González (2001) for significant differences in the contents of protein, lipids, carbohydrates, and energy in the gonad, in response to protein dietary content. Procedures available in STATISTICA 6® and G*Power 2 (StatSoft®) were used to conduct the analyses. 3. Results No significant differences were found in survival (78.6–84.5%), final weight (41.0–43.1g), and fecundity (8.5–9.2 eggs/g female) of female redclaw crayfish using the different levels of dietary protein (Table 2). A significant regression analysis showed a satisfactory fit of the quadratic equation to percentage of spawning females. The protein level yielding the maximum percentage was 30% (Fig. 1). The percentage of spawning females ranged from 19.7 to 27.3%. There were significant differences in morphological features of eggs. Egg area ranged from 3.60 to 3.90 mm2, volume from 34.5 to 39.3 mm3, weight from 4.68 to 5.44 mg, and diameter from 2.17 to 2.27 mm. Maximum values were consistently present in crayfish fed the 32% protein diet (Fig. 2). Energy, mean protein, lipid, or carbohydrate contents did not differ significantly relative to dietary protein level. High statistical power (> 0.95) indicated that protein, lipid, and carbohydrate levels in the egg were unaffected by the dietary level of protein (Table 3).
4. Discussion Dietary protein influenced the proportion of redclaw crayfish spawners and the size and weight of their eggs. The role of dietary nutrients for broodstock, such as protein, has been studied for other crustaceans (Tacon and Cowey, 1985; Watanabe et al., 1985). Gonad maturation, fecundity, and quality of both eggs and juveniles of aquatic organisms are influenced by broodstock nutrition (Bromage, 1995; Rodríguez-González, 2001; Wouters et al., 2001). Protein is a structural, functional, and energy constituent of tissues, and plays an important role in spawning, fertilization, and normal development of the embryo in decapods (Harrison, 1990; Wouters et al., 2001; García-Guerrero et al., 2003). Several authors (i.e. Yeh and Rouse, 1994; Barki et al., 1997; Abdu et al., 2001; McPhee et al., 2004) have presented spawning rates for the species. The values reported in the present study are within the range reported, and comparable to those indicated by Yeh and Rouse (1995), for similar density, sex ratio and water temperature. This study shows that a dietary protein level of about 30% maximizes the size of the spawning population. This suggests that such protein level is optimal to meet nutritional requirements for reproductive females. While there are no previous studies determining optimal dietary protein requirement for spawning female redclaw crayfish, these are similar to recommendations made for growout of pre-adults (27%
35 30 25 Spawning (%)
Table 2 Mean final weight, survival and fecundity of female Cherax quadricarinatus fed diets containing different dietary levels of protein (22, 27, 32, and 37% CP) for 75 days
415
20 15 10 2
s=-0.12CP +7.17CP-80.97
5
2
R = 0.96 0 17
22
27
32
37
42
% Crude Protein
Fig. 1. Mean spawning (s) percentage of Cherax quadricarinatus females fed with four crude protein (CP) levels (22, 27, 32, and 37%) for 75 days. The crude protein yielding the maximum percentage of spawning was calculated from regression coefficients as: 7.17/ (2 × 0.12) = 30 (dashed line).
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A
B 2.45
6.0
2.00
2.75
a
a 2.70
2.30 2.25
4.5 a
2.20
ab
4.0
b
Wet weight Mean diameter
b
22
27
32
37
b
b
1.90
2.60 2.55
1.85
a
2.50 2.45 2.40
2.10
2.35
1.80
Major diameter
1.75
b
b
2.15
ab
Minor diameter
3.5 17
Major diameter (mm)
Mean diameter (mm)
Wet weight (mg)
2.35
b b
5.0
1.95
ab 2.65
Minor diameter (mm)
2.40 ab
5.5
1.70 17
42
22
27
% Crude Protein
32
37
42
% Crude Protein
C 46
4.1
a
4.0
44
3.9
ab
2
Area (mm )
3
b
b
40
3.7 3.6
38
a
3.5
Volume (mm )
42 3.8
36
ab
3.4 3.3
Volume
b
3.2 17
22
34
Area
b
32 27
32
37
42
% Crude Protein
Fig. 2. Mean wet weight, diameter (A), major and minor diameter (B), area and volume (C) of eggs from female Cherax quadricarinatus fed diets containing different dietary protein levels (22, 27, 32, and 37%).
crude protein; Cortés-Jacinto et al., 2004) and juveniles (32% crude protein; Cortés-Jacinto et al., 2005). The growth rates in our trial were smaller than those reported by Cortés-Jacinto et al. (2003), Thompson et al. (2003),
and Thompson et al. (2005) for juveniles. However, they were comparable to those reported by Cortés-Jacinto et al. (2004), for similar sized organisms. The different protein levels used in this experiment yielded a
Table 3 Mean biochemical composition (n = 12) of eggs from Cherax quadricarinatus females fed diets containing different levels of dietary protein (22, 27, 32, and 37%) Diet (%CP)
Protein (μg/egg)
Lipid (μg/egg)
Carbohydrate (μg/egg)
Energy (J/egg)
22 27 32 37 Statistical power
2107.70 ± 544.3 2369.730 ± 320.3 2176.660 ± 530.4 2238.510 ± 331.3 0.96
440.260 ± 80.1 408.430 ± 93.3 422.150 ± 74.6 463.080 ± 94.1 0.99
70.690 ± 6.9 69.230 ± 10.7 78.600 ± 10.1 75.530 ± 12.2 0.98
53.700 ± 4.3 57.760 ± 7.4 55.590 ± 9.2 55.220 ± 6.4 0.99
No significant differences were found (P > 0.05).
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fecundity value (8.5–9.2 eggs/g) similar to that reported by Yeh and Rouse (1994) (7.8–10 egg/g female). Egg quality is defined as the ability of an egg to be fertilized and undergo development (Holcomb et al., 2004). Morphological features of eggs, hatching rates, biochemical composition, and levels of specific nutrients are generally deemed to be good indicators of egg quality (Clarke et al., 1990; Lavens et al., 1991; Watanabe et al., 1991; García-Ulloa et al., 2004). In our study, mean egg diameter, volume, area, and weight were higher for the 32% crude protein diet. This value closely approximates the one calculated to maximize the size of spawning population, suggesting that 32% crude protein in the diet is optimal for obtaining high quality eggs. Information on morphological features of C. quadricarinatus eggs is scarce, however, the mean newly laid egg size obtained in our study (2.60 mm) coincides with the size of oocytes before laying (2.55 mm) reported by Sagi et al. (1996). We found that protein, lipid, carbohydrate, and energy in eggs were not affected by dietary protein level and that, for the conditions studied, biochemical composition cannot be used as an indicator of egg quality. Two factors may be responsible for this response. García-Guerrero et al. (2003) stated that the biochemical composition of eggs in C. quadricarinatus is dependent on the nutritional status of the female. The reproductive strategy of the species also plays a role. The redclaw crayfish has relatively low fecundity and exhibits maternal care (Holdich, 2002). Under this strategy, it is hypothesized the reproductive effort of the female is aimed at guaranteeing uniform egg quality and reproductive success by using different nutrient sources, and that spawning occurs only after a balanced and uniform biochemical composition of eggs is obtained. From the results in the present study, it can be concluded that a dietary protein content of 32% is recommended for reproduction of female redclaw C. quadricarinatus. However the actual value may vary according to dietary ingredient composition, digestibility (Campaña-Torres et al., 2005) and methodology used for protein determination. This information should contribute to the development of adequately balanced practical diets for female redclaw crayfish broodstock. Acknowledgements The authors thank Beatriz Romero, Francisco Encarnación, and Ismael Valdivia for assistance during the trials, and Sonia Rocha and Dolores Rondero for the biochemical analyses. The editor at CIBNOR provided
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