Effect of protein source ratio and protein concentration in prepared diets on gonad yield and quality of the green sea urchin, Strongylocentrotus droebachiensis

Aquaculture 214 (2002) 307 – 332 www.elsevier.com/locate/aqua-online

Effect of protein source ratio and protein concentration in prepared diets on gonad yield and quality of the green sea urchin, Strongylocentrotus droebachiensis Christopher M. Pearce a,*, Tara L. Daggett b, Shawn M.C. Robinson c a

Applied Aquaculture Section, Pacific Biological Station, Department of Fisheries and Oceans, 3190 Hammond Bay Road, Nanaimo, British Columbia, Canada V9T 6N7 b Ross Island Salmon Ltd., P.O. Box 1304, Grand Manan, New Brunswick, Canada E5G 4M9 c Applied Aquaculture Section, St. Andrews Biological Station, Department of Fisheries and Oceans, 531 Brandy Cove Road, St. Andrews, New Brunswick, Canada E5B 2L9 Received 26 October 2001; received in revised form 28 January 2002; accepted 28 January 2002

Abstract Adult green sea urchins (Strongylocentrotus droebachiensis) were collected from the wild and held in laboratory tanks where they were fed a number of prepared diets or a control of kelp (Laminaria longicruris and/or L. digitata) for a period of 12 weeks. Twelve different prepared diets were formulated in order to examine two experimental factors: (1) protein concentration at three different levels [19%, 24%, and 29% (percent dry weight of all dietary components)] and (2) protein source ratio at four different levels [rockweed meal, wheat meal, corn meal, soybean meal, fish meal in the following four ratios: 0:30:30:25:15, 15:20:20:35:10, 30:10:10:45:5, 45:0:0:55:0, respectively (percent dry weight of all meals)]. Each concentration was present at each source ratio in a completely crossed experimental design. Gonad colour, percent gonad water, and percent gonad yield of experimental urchins were determined approximately every week while gonad texture, firmness, and taste were subjectively evaluated at the end of the experiment. Results were contrasted with those of wild specimens collected from the source population at weeks 0 and 12 of the experiment. After 12 weeks, all prepared diets produced significantly higher percent gonad yields than kelp-fed urchins or wild controls, the best-prepared diet giving a mean weekly increase of 1.3% in percent yield. Percent gonad yield was not significantly affected by protein concentration, but was significantly affected by protein source ratio; the 15:20:20:35:10 diet gave significantly higher yields than the 0:30:30:25:15 or 45:0:0:55:0 diets (30:10:10:45:5 being intermediate). Gonad colour of urchins fed prepared diets

* Corresponding author. Tel.: +1-250-756-7000; fax: +1-250-756-7053. E-mail address: [email protected] (C.M. Pearce). 0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 0 4 1 - 8

308

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

was, generally, pale yellow/orange to yellow-brown/orange-brown at the end of the experiment and not significantly different from kelp-fed urchins or wild controls. Gonad colour was not significantly affected by protein source ratio, but was significantly affected by protein concentration; the 19% and 24% levels produced significantly better coloured gonads than the 29% level. At the end of the experiment, gonad texture of urchins fed prepared diets ranged from smooth to very smooth with distinct gonad segment halves while gonad firmness ranged from firm to very firm. Gonad texture and firmness of prepared diet treatments did not differ significantly from kelp-fed urchins or wild controls and neither protein concentration nor protein source ratio significantly affected gonad texture or firmness. At the end of the experiment, gonad taste of urchins fed prepared diets ranged from bitter to sweet with all prepared diets producing significantly worse tasting gonads than the kelp-fed urchins. Gonad taste was not significantly affected by protein source ratio nor protein concentration, but there was a general trend of worsening taste with increasing protein levels. Based on these results, a 19% protein concentration and a 15:20:20:35:10 (rockweed meal/wheat meal/corn meal/soybean meal/fish meal) protein source ratio are recommended for further dietary research. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Gonad; Protein; Prepared diet; Roe quality; Urchin; Strongylocentrotus droebachiensis

1. Introduction Sea urchin gonads or ‘‘roe’’ fetch high prices on the Japanese seafood market—as much as US$400 per kg depending on the time of year and urchin species. These high prices, coupled with a fairly stable market demand and the recent decline in natural fisheries in the major sea urchin producing nations (Keesing and Hall, 1998), has created much interest in the development of sea urchin aquaculture. While the major species of sea urchins being considered for culture are predominantly grazers of macroalgae, the use of macrophytes for large-scale commercial culture of urchins (either full life-cycle production or gonad enhancement of wild-caught individuals) is unlikely to be commercially viable due to a number of reasons including: (1) limited natural resources of suitable macroalgal species; (2) temporal variation in quantity and/or quality of algae; (3) restricted harvesting of macroalgal stocks in some areas; (4) conflict with other marine users (e.g. fishers, land owners); (5) expense associated with collecting large quantities of macroalgae; and (6) difficulty in storing commercial-scale quantities of algae. The development of a suitable prepared diet for the culture of sea urchins has thus been a major scientific and industrial research focus in recent years. Gonad production of wild-caught sea urchins has been achieved using prepared diets in a number of different sea urchin species including: Evechinus chloroticus (Barker et al., 1998; Goebel and Barker, 1998), Loxechinus albus (Lawrence et al., 1997; Olave et al., 2001), Paracentrotus lividus (Lawrence et al., 1992; Fernandez and Boudouresque, 1998, 2000; Fernandez and Pergent, 1998; Spirlet et al., 2000), Pseudocentrotus depressus (Unuma et al., 1999; Akiyama et al., 2001), Strongylocentrotus droebachiensis (de JongWestman et al., 1995a; Klinger et al., 1997; Walker and Lesser, 1998; Ha´vardsson et al., 1999; Pearce et al., 2002; Robinson et al., in press), S. franciscanus (McBride et al., 1997, 1999), and S. intermedius (Levin and Naidenko, 1987). While the use of prepared

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

309

diets to increase urchin gonad yield has been substantially documented, the quality of gonads from individuals fed these diets has received considerably less attention (but see Motnikar et al., 1997; Goebel and Barker, 1998; Watts et al., 1998; Pearce et al., 2002; Robinson et al., in press). Since the marketability and price of urchin roe are inextricably linked to quality factors such as gonad colour, firmness, and taste, gonad quality is at least as important as gonad yield in the commercial roe industry. One of the primary benefits of using a prepared diet in sea urchin nutritional studies from a scientific research perspective is that the diet formulation is known and can be replicated. Dietary ingredients can therefore be manipulated and the effect of such changes on feeding/production documented. Protein is one such dietary component that has been manipulated in urchin feeding trials with prepared diets. Generally, the focus has been on determining the effect of varying protein concentration or protein source on ingestion rate, absorption efficiency, feed digestibility, assimilation efficiency, somatic growth rate, biochemical composition of body parts, and/or egg/larval quality (Lares and McClintock, 1991; Lawrence et al., 1992; Fernandez and Caltagirone, 1994; Klinger et al., 1994; de Jong-Westman et al., 1995b; Akiyama et al., 1997, 2001; Fernandez, 1997; McBride et al., 1998; Fernandez and Boudouresque, 1998, 2000; Kennedy et al., 2001) and surprisingly little information is available on the effect of protein concentration or source on the production of gonads. The objective of the present study was to determine the effect of varying protein concentration and protein source ratio on the yield and quality (i.e. colour, texture, firmness, taste) of gonads of adult green sea urchins. Three different protein concentrations [19%, 24%, 29% (percent dry weight of all ingredients)] were tested, each at four different ratios of the following protein meals: rockweed meal, wheat meal, corn meal, soybean meal, fish meal [ratio: 0:30:30:25:15, 15:20:20:35:10, 30:10:10:45:5, 45:0:0:55:0, respectively (percent dry weight of all meals)].

2. Materials and methods 2.1. Sea urchin collection and maintenance Adult sea urchins, S. droebachiensis, of test diameter 59.6 F 4.1 mm (mean F S.D., n = 30) were collected by scuba divers off Bancroft Point, Grand Manan Island, New Brunswick (44j43VN, 66j44VW) on a rocky, cobble bottom at a depth of approximately 10 m (high tide depth) on July 22– 23, 1998. They were transported to the laboratory within 3 h of collection in plastic tote boxes with ambient seawater. They were then placed in white plastic tanks (L
W
H: 50
50
28 cm) at an initial stocking density of 120 urchins tank 1 or f 180 kg m 3 or f 15 kg m 2 (mean weight of urchins F S.D.: 90.3 F 16.0 g, n = 30). These tanks were supplied with flow-through, ambient seawater at a rate of f 5 l min 1 and were equipped with a double stand-pipe (ID of outer pipe: 35 mm; ID of inner pipe: 18 mm) that was rigged so that seawater that flowed into the tanks at the top was forced out at the bottom. Urchins were allowed to adjust to laboratory conditions for 12 –13 days prior to experimentation. During that time, the urchins were starved to standardise the relative hunger levels of the animals and

310

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

to allow any critically injured individuals to be culled from the experimental group and replaced. Lighting was provided by overhead fluorescent lights that were controlled by an astronomic time switch (Model EL71, Paragon Electrical Products, Maple Chase, Downers Grove, IL, USA). On August 10, 1998, during the first week of the experiment, the lights were set for a photo-period corresponding to the calendar date of October 1, 1998 using published sunrise and sunset times. Experimental lighting was phase-shifted 2 months ahead of calendar date and ambient photo-period in order to mimic day lengths during the time of the year when natural sea urchin populations are increasing gonadal reserves. Temperature was automatically recorded in a header tank every 15 min by a temperature data logger and gradually decreased during the experimental period (max: 15.6 jC, min: 8.1 jC, mean F S.D.: 10.8 F 1.2 jC, n = 5438). 2.2. Diet preparation Pork gelatin (purity level or bloom factor: 175) was obtained from CSP Foods (Moncton, New Brunswick, Canada) and corn oil/corn starch from a local supermarket. All other ingredients were purchased from Shur-Gain, Maple Leaf Foods (Truro, Nova Scotia, Canada), a local animal-feed supplier. The basic diet formulation was based on that used by Pearce et al., 2002 using 5% pork gelatin as the binder (this was found to be the best of a number of different binders tested in terms of maintaining pellet stability). Slight modifications were made to the basal diet due to the findings of Pearce et al., 2002 and included: (1) decrease in concentration of fish meal, (2) deletion of fish oil, and (3) increase in concentration of h-carotene from 0.2 to 0.3 mg kg 1 dry weight of feed. A small Hobart mixer/grinder (Hobart, Troy, OH, USA) was used to mix the dry ingredients with hot freshwater ( f 100 jC) and to extrude a moist pellet (diameter: 5/16 in. or f 7.9 mm). These pellets were then air-dried outdoors on warm sunny days and later stored in a refrigerator at f 3– 5 jC until further use. Three separate batches of diets were made during the experiment. Twelve different prepared diets were formulated in order to examine two experimental factors: (1) protein concentration at three different levels [19%, 24%, and 29% (percent dry weight of all dietary components)] and (2) protein source ratio at four different levels [rockweed meal (Ascophyllum nodosum), wheat meal, corn meal, soybean meal, fish meal in the following ratios: 0:30:30:25:15, 15:20:20:35:10, 30:10:10:45:5, 45:0:0:55:0, respectively (percent dry weight of all meals)]. Protein concentration was calculated based on the protein levels within each dietary ingredient as determined by the feed supplier or manufacturer. The specific protein source ratios were chosen, in part, to test the effect of varying rockweed meal and fish meal concentrations on gonad production and quality. Fish meals and oils, while shown to promote excellent gonad growth in sea urchins, usually impart a bitterness or oiliness to the gonads (Hoshikawa et al., 1998; Pearce et al., 2002). The concentration of fish meal in the prepared diets in the present study was generally kept below that in the diet used by Pearce et al., 2002 to reduce the potential of imparting a bitter taste to the gonads. The protein source ratios also reflect the limitations of producing diets containing a specific protein concentration. A variety of different meals was utilized in the diets in order to provide a balanced amino acid profile.

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

311

Each protein concentration was tested at each protein source ratio in a totally crossed experimental design. For brevity, treatment names are abbreviated using the rockweed meal and protein concentrations, respectively (e.g. the ratio 45:0:0:55:0 at 19% protein is abbreviated to 45:19). Changes in concentration and source ratio of meals was accomplished without altering the balance of important nutritional elements such as lipid, vitamins, and minerals by modifying the levels of corn starch, which was added to each diet as a filler (as in de Jong-Westman et al., 1995a). Ingredients and protein levels in the various prepared diets are shown in Table 1. 2.3. Experimental protocols The experiment was conducted for 12 weeks (August 4– October 27, 1998). In addition to the 12 prepared diet treatments (see Diet preparation), there was a control treatment of kelp (blades of Laminaria longicruris and/or L. digitata). Three replicate tanks were established for each of the 13 treatments, each replicate having 120 urchins at the beginning of the experiment and being placed in a separate group in a completely randomized block design. Sea urchins were fed twice a week (usually Monday and Friday) at a rate of 0.9 –1.2% body weight day 1 of prepared diet and 2.6 –3.5% body weight day 1 of kelp, the rate depending on the interval between feedings. These rates were at or above satiation levels and were based on observations by Pearce et al. (2002). Urchins were hand-fed and attempts were made to ensure that all urchins had equal access to feed. Tanks were cleaned twice a week before feeding by removing the stand pipes, allowing the tanks to drain, and washing the uneaten feed and faecal material out of the tanks with ambient seawater. Dead urchins were noted and removed from the tanks on approximately a daily basis, but not replaced. A random sample of 30 urchins, chosen from extra individuals that were not part of the study, was taken at the beginning of the experiment to assess initial gonad yield and colour. Yield and colour were then assessed every week, except for the fifth and sixth weeks, for the 12-week duration of the experiment by randomly sampling 10 urchins from each replicate tank. Feeding rates were adjusted for sampled individuals but not dead individuals, since percent mortality during the experiment was extremely low [ < 6.5% per 12 weeks in all treatments (see Urchin mortality)]. Wild samples from the source population (i.e. Bancroft Point) were collected at the end of the experiment for assessment of yield and quality. Sampled urchins were vigorously shaken to remove excess external water and their test diameters and weight measured using digital calipers and a digital balance, respectively. They were then cracked open, thoroughly drained of internal fluid, and re-weighed. The gonads were scooped out of the urchin tests using a uni spoon, cleaned in seawater, and gently shaken using forceps to remove as much water as possible, but not blotted dry. The gonads were then placed in pre-weighed aluminum pans, weighed, assessed for colour, dried to a constant weight in a 70 jC oven (generally a minimum of 48 h), and reweighed. Colour was assessed using 79 different paint card samples (Home Hardware, Beauti-Tone) that were later converted to a rating of 1 –4 (see scale below). At the end of the experiment, gonad colour was also subjectively rated without using the paint samples

312

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

and gonad texture and firmness were evaluated using the 10 randomly sampled individuals from each replicate tank. Gonad colour was always assessed under standardised light conditions [i.e. f 50 cm away from a single-point, artificial light source (20 W Sylvania Table 1 Ingredients and protein levels used in diets Ingredient (dry weight % of meals)

Dry weight (% of total) [protein (% of total)] 15% Diet

20% Diet

25% Diet

Protein ratio: 0:30:30:25:15 Rockweed meal (0%) Wheat meal (30%) Corn meal (30%) Soybean meal (25%) Fish meal (15%) Corn starch (unmodified) Gelatin Other ingredientsa Total

0.000 [0.00] 15.712 [2.04] 15.708 [1.18] 13.094 [6.29] 7.855 [5.50] 37.336 [0.10] 5.000 [4.29] 5.295 [0.00] 100.000 [19.39]

0.000 [0.00] 20.942 [2.72] 20.942 [1.57] 17.451 [8.38] 10.470 [7.33] 19.900 [0.05] 5.000 [4.29] 5.295 [0.00] 100.000 [24.34]

0.000 26.183 26.183 21.818 13.088 2.433 5.000 5.295 100.000

[0.00] [3.40] [1.96] [10.47] [9.16] [0.01] [4.29] [0.00] [29.30]

Protein ratio: 15:20:20:35:10 Rockweed meal (15%) Wheat meal (20%) Corn meal (20%) Soybean meal (35%) Fish meal (10%) Corn starch (unmodified) Gelatin Other ingredientsa Total

7.768 10.361 10.358 18.129 5.182 37.907 5.000 5.295 100.000

[0.54] [1.35] [0.78] [8.70] [3.63] [0.10] [4.29] [0.00] [19.38]

10.359 13.816 13.817 24.180 6.909 20.624 5.000 5.295 100.000

[0.73] [1.80] [1.04] [11.61] [4.84] [0.05] [4.29] [0.00] [24.34]

12.955 17.274 17.275 30.232 8.633 3.336 5.000 5.295 100.000

[0.91] [2.25] [1.30] [14.51] [6.04] [0.01] [4.29] [0.00] [29.30]

Protein ratio: 30:10:10:45:5 Rockweed meal (30%) Wheat meal (10%) Corn meal (10%) Soybean meal (45%) Fish meal (5%) Corn starch (unmodified) Gelatin Other ingredientsa Total

15.384 5.130 5.127 23.079 2.562 38.423 5.000 5.295 100.000

[1.08] [0.67] [0.38] [11.08] [1.79] [0.10] [4.29] [0.00] [19.39]

20.509 6.837 6.833 30.762 3.421 21.343 5.000 5.295 100.000

[1.44] [0.89] [0.51] [14.77] [2.39] [0.06] [4.29] [0.00] [24.34]

25.646 8.545 8.547 38.466 4.276 4.225 5.000 5.295 100.000

[1.80] [1.11] [0.64] [18.46] [2.99] [0.01] [4.29] [0.00] [29.30]

Protein ratio: 45:0:0:55:0 Rockweed meal (45%) Wheat meal (0%) Corn meal (0%) Soybean meal (55%) Fish meal (0%) Corn starch (unmodified) Gelatin Other ingredientsa Total

22.839 0.000 0.000 27.919 0.000 38.947 5.000 5.295 100.000

[1.60] [0.00] [0.00] [13.40] [0.00] [0.10] [4.29] [0.00] [19.39]

30.455 0.000 0.000 37.225 0.000 22.025 5.000 5.295 100.000

[2.13] [0.00] [0.00] [17.87] [0.00] [0.06] [4.29] [0.00] [24.34]

38.077 0.000 0.000 46.530 0.000 5.098 5.000 5.295 100.000

[2.67] [0.00] [0.00] [22.33] [0.00] [0.01] [4.29] [0.00] [29.30]

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

313

Cool White flourescent light) with no natural lighting]. Gonad taste was also assessed at the end of the experiment by randomly sampling three urchins from each replicate tank; each urchin being sampled by two independent tasters. Gonad colour, texture, firmness, and taste were also evaluated for wild urchins collected from Bancroft Point at the end of the experiment. Gonad characteristics were quantified as follows Gonad Yield ð%Þ ¼ wet gonad weight=whole urchin weight
100 Gonad Water ð%Þ ¼ ½ðwet gonad weight  dry gonad weightÞ =wet gonad weight
100 Gonad Colour: subjectively by eye with or without paint samples (rating 1 –4) 1 = bright yellow or orange (equivalent to Grade A in commercial roe industry) 2 = paler yellow or orange, mustard (Grade A or Grade B) 3 = yellow-brown, orange-brown, red-brown, cream (Grade B or Grade C) 4 = any other colour (e.g. dark brown, grey) (Grade C) Gonad Texture: subjectively by eye (rating 1– 4) 1 = two distinct gonad segment halves, very smooth 2 = two distinct gonad segment halves, smooth (distinction and smoothness < 1) 3 = distinction of gonad segment halves possible but < 2, rough/granular 4 = distinction of gonad segment halves not possible, rough/granular Gonad Firmness: subjectively by eye (rating 1 –4) 1 = very firm 2 = firm 3 = soft 4 = very soft

Notes to Table 1: a Other ingredients include the following that were added in the same proportions to each diet (% dry weight, % protein): dicalcium phosphate (1.800, 0.00), ethoxyquin (0.200, 0.00), vitamin C (Stay C) (0.080, 0.00), vitamin pre-mixb (0.100, 0.00), mineral pre-mixc (0.100, 0.00), Rovimix h-carotene 0.2%d (0.015, 0.00), lecithin (1.000, 0.00), and corn oil (2.000, 0.00). b Contains ground wheat, vitamin E, vitamin C (Stay C), inositol, ethoxyquin, vitamin D3, niacin, calcium pantothenate, vitamin K, soybean oil, vitamin B12, biotin, riboflavin, pyridoxine, thiamine, vitamin A, folic acid. Levels are proprietary information. c Contains ground wheat, manganese sulphate, iron sulphate, zinc sulphate, soybean oil, calcium iodate, selenium selenate, copper chloride. Levels are proprietary information. d Contains 98% wheat middlings and 0.2% h-carotene. Therefore, the actual h-carotene concentration in diet is 0.00003% or 0.3 mg kg 1 dry weight of feed.

314

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

Gonad Taste: subjectively by two independent tasters (rating 1– 6) 1 = excellent (very sweet) 2 = very good (very sweet, but < 1) 3 = good (sweet) 4 = satisfactory (bland: not sweet, not bitter) 5 = poor (bitter) 6 = very poor (very bitter)

2.4. Statistics For percent gonad yield, percent gonad water, gonad colour, gonad texture, and gonad firmness of experimental animals, a mean was calculated using the 10 urchins sampled from each replicate tank (for the wild urchins sampled at weeks 0 and 12, thirty individuals were randomly placed in three groups of 10 in order to obtain three mean values). This mean ‘‘tank’’ value was then used in subsequent statistical analyses (n = 3). For gonad taste, a mean value was calculated for each urchin using the two independent observations and this value was then used in the calculation of mean tank values. These tank values were then used in statistical analyses (n = 3). For the various gonad characteristics, two-way ANOVAs (completely randomized block design with tank as the blocking factor) were used to determine the significance of the differences among all treatments at the end of the experiment, including the kelp-fed urchins and wild samples taken at the beginning and end of the experiment. To examine the combined effects of protein concentration and protein source ratio more closely, three-way ANOVAs (concentration, source ratio, and tank as blocking factor) were conducted on gonad yield, colour, texture, firmness, and taste at the end of the experiment. Four-way, repeated measures ANOVAs (concentration, source ratio, time, and tank as blocking factor) were utilized to assess the effect of protein concentration and protein source ratio on percent gonad yield, percent gonad water, and gonad colour over time. In multi-way ANOVAs with significant interaction terms, the effect of one main factor was examined within each level of the other main factor with one-way ANOVAs using the Bonferroni procedure for protection of an overall P of 0.05. Where significant P-values were generated in ANOVAs, Fisher’s LSD post-hoc comparison tests were used to evaluate pair-wise means. Probability plots were used to confirm that data were normally distributed and Cochran’s tests ( P < 0.01) utilized to verify that variances were homogeneous.

3. Results 3.1. Diet stability Stability tests were run as described in Pearce et al. (2002) to determine if protein concentration or source ratio affected the coherence of the pelletized diet. All protein concentration/source ratio combinations produced pellets that maintained their consistency

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

315

for a period of 6 days (maximum length of stability trials) in running seawater. There was no significant difference among the various treatments in regards to pellet consistency. 3.2. Urchin mortality Cumulative percent mortality over the 12-week experimental period ranged from a low of 0.6 F 0.3% (mean F S.E.) for 15:29 and 45:19 to a high of 6.1 F 3.1% for 0:19. All treatments except for 0:19 had less than 4% cumulative mortality and there was no significant difference among treatment means (Table 2). 3.3. Gonad yield Percent gonad yield grew from 9.7% at the beginning of the experiment to at least 20.9% in all prepared diet treatments with percent gonad yield increases per week ranging from a low of 0.9% for 0:24 to a high of 1.3% for 15:29 (Fig. 1). Mean percent gonad yields for the feeding treatments at the end of the 12-week experimental period and the wild samples collected at the beginning and end of the experiment differed significantly (Table 2). All prepared diets produced significantly greater percent gonad yields (range: 20.9 F 1.2% to 25.5 F 1.1%) than those fed kelp (15.3 F 0.4%) or those sampled from the wild either at the beginning (9.7 F 0.3%) or end (11.7 F 0.9%) of the experiment, while the kelp control produced significantly greater percent gonad yield than either of the wild samples (Fig. 2A). There was no significant difference in percent gonad yield between wild samples collected at the beginning or end of the experiment (Fig. 2A). Among the prepared diets, 15:29 gave the highest percent gonad yield (25.5 F 1.1%) which was significantly greater than 0:19 (21.9 F 0.2%), 0:24 (20.9 F 1.2%), 0:29 (22.5 F 0.7%), 30:19 (22.1 F 1.6%), 45:19 (22.2 F 0.4%), and 45:24 (22.9 F 0.9%) (Fig. 2A). The 15:24 (25.0 F 0.9%) and 30:24 (24.9 F 2.2%) diets gave the second and third highest percent gonad yields, respectively, and were both significantly greater than the 0:19, 0:24, 30:19, and 45:19 diets (Fig. 2A). There was only one other significant pair-wise comparison among the prepared diets: 15:19 (24.1 F 0.5%) had greater percent gonad yield than 0:24 (Fig. 2A). A three-way ANOVA, examining the effects of protein concentration and protein source ratio on percent gonad yield at the end of the experiment, revealed a significant effect of source ratio (Table 2). The effect of source ratio was consistent across all protein concentrations leading to a non-significant interaction effect (Table 2). The 15:20:20:35:10 diet produced significantly greater percent gonad yield than either the 0:30:30:25:15 or 45:0:0:55:0 diets (Fig. 3A). The 0:30:30:25:15, 30:10:10:45:5, and 45:0:0:55:0 diets did not differ significantly from one another (Fig. 3A). There was no significant effect of protein concentration on percent yield (Table 2). A four-way repeated ANOVA, examining the effects of protein concentration and protein source ratio on percent gonad yield over time, revealed significant effects of source ratio and time, but no significant effect of protein concentration (Table 3). The effect of protein source ratio on percent gonad yield varied with time as is evident from the significant interaction between the two factors (Table 3). For the first 7 weeks of the experiment, percent gonad yield did not vary significantly among the protein source ratios

316

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

Table 2 ANOVA results (two-way and three-way) on cumulative percent mortality, percent gonad yield, gonad colour, texture, firmness, and taste at the end of the 12-week experiment Data set/analysis

Source

SS

df

F-ratio

P-value

Mortality/two-way ANOVA

Treatment Block Error Treatment Block Error Source ratio (S) Concentration (C) S
C Block Error Treatment Block Error Source ratio (S) Concentration (C) S
C Block Error Treatment Block Error Source ratio (S) Concentration (C) S
C Block Error Treatment Block Error Source ratio (S) Concentration (C) S
C Block Error Treatment Block Error Source ratio (S) Concentration (C) S
C Block Error Treatment Block Error

84.65 0.96 129.13 981.73 21.17 70.02 45.76 7.13 13.37 20.48 64.74 1.48 0.04 1.71 0.09 0.80 0.04 0.00 1.32 0.98 0.03 1.07 0.02 0.07 0.09 0.03 0.97 0.98 0.14 1.26 0.08 0.00 0.57 0.10 1.18 2.51 0.12 3.64 0.04 0.27 0.58 0.15 3.47 19.86 2.20 13.24

12 2 24 14 2 28 3 2 6 2 22 14 2 28 3 2 6 2 22 13 2 26 3 2 6 2 22 13 2 26 3 2 6 2 22 13 2 26 3 2 6 2 22 13 2 26

1.31 0.09

> 0.1 NS > 0.5 NS

Gonad Yield/two-way ANOVA

Gonad Yield/three-way ANOVA

Gonad Colour/two-way ANOVA (with paint samples) Gonad Colour/three-way ANOVA (with paint samples)

Gonad Colour/two-way ANOVA (without paint samples) Gonad Colour/three-way ANOVA (without paint samples)

Gonad Texture/two-way ANOVA

Gonad Texture/three-way ANOVA

Gonad Firmness/two-way ANOVA

Gonad Firmness/three-way ANOVA

Gonad Taste/two-way ANOVA

28.04 4.23

< 0.001**** < 0.05*

5.18 1.21 0.76 3.48

< 0.01** > 0.1 NS > 0.5 NS < 0.05*

1.73 0.33

> 0.1 NS > 0.5 NS

0.50 6.67 0.12 0.02

> 0.5 NS < 0.005*** > 0.5 NS > 0.5 NS

1.82 0.35

>0.05 NS >0.5 NS

0.18 0.84 0.35 0.31

> 0.5 > 0.1 > 0.5 > 0.5

1.55 1.48

> 0.1 NS > 0.1 NS

0.52 0.02 1.76 0.95

> 0.5 > 0.5 > 0.1 > 0.1

1.38 0.43

> 0.1 NS > 0.5 NS

0.08 0.86 0.62 0.48

> 0.5 > 0.1 > 0.5 > 0.5

3.00 2.16

< 0.01** > 0.1 NS

NS NS NS NS

NS NS NS NS

NS NS NS NS

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

317

Table 2 (continued) Data set/analysis

Source

Gonad Taste/three-way ANOVA

Source ratio (S) Concentration (C) S
C Block Error

SS 0.58 2.60 1.46 1.49 12.46

df

F-ratio

3 2 6 2 22

0.34 2.30 0.43 1.32

P-value >0.5 >0.1 >0.5 >0.1

NS NS NS NS

(Fig. 4). In weeks 8 through 12, the 15:20:20:35:10 diet produced greater percent gonad yield than any of the other ratios; this was significantly greater than the 0:30:30:25:15 diet in weeks 8, 10, 11, and 12 and significantly greater than the 45:0:0:55:0 diet in weeks 8 and 12, but never significantly different from the 30:10:10:45:5 diet (Fig. 4). The 0:30:30:25:15 diet always produced the lowest percent gonad yield of any protein source ratio, although not all pair-wise comparisons were significantly different (Fig. 4). 3.4. Gonad colour Mean gonad colour ratings for all treatments fluctuated between 2 and 3 for the experimental period (Fig. 5). At the end of the experiment, mean colour of urchin gonads from prepared diet treatments varied between 2.1 F 0.2 (0:19, 15:19) and 2.6 F 0.1 (30:29) for ratings done with paint samples and between 2.5 F 0.1 (0:19, 15:19, 15:24, 30:19, 45:24) and 2.7 F 0.1 (0:24, 30:29) for ratings done without paint samples (Fig. 2B,C). While mean colour ratings of kelp (2.1 F 0.2 with paint samples, 2.3 F 0.1 without paint samples), wild 0-week (2.1 F 0.1 with paint samples), and wild 12-week (2.0 F 0.1 with paint samples, 2.1 F 0.1 without paint samples) treatments were slightly better than any prepared diet at the end of the experiment, there was no significant difference among any of the treatments in either data set (Table 2, Fig. 2B,C). A three-way ANOVA, examining the effects of protein concentration and protein source ratio on gonad colour ratings done with paint samples at the end of the experiment, showed a significant concentration effect, but non-significant source ratio and interaction effects (Table 2). The 19% and 24% protein levels produced significantly better gonad colour than the 29% level (Fig. 3B). There was no significant difference in gonad colour, however, between the 19% and 24% protein concentrations (Fig. 3B). Unlike the paint sample data, a three-way ANOVA conducted on the colour ratings done without paint samples at the end of the experiment did not detect a significant effect of protein concentration (Table 2). While not significantly different, the 19% and 24% protein levels did, generally, produce slightly better gonad colour than the 29% level (Fig. 3C). As with the paint sample data, effects of protein source ratio and the interaction between concentration and source ratio were both non-significant for the colour ratings done without paint samples (Table 2). A four-way repeated ANOVA, examining the effects of protein concentration and protein source ratio on colour ratings done with paint samples over the 12-week experimental period, revealed a significant effect of time, but non-significant effects of

318 C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

Fig. 1. Mean percent gonad yield in the various prepared diet treatments at each sampling period. Error bars are S.E. and n = 3.

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332 319

Fig. 2. Mean percent gonad yield (A), mean gonad colour rating done with paint samples (B), mean gonad colour rating done without paint samples (C), mean gonad texture rating (D), mean gonad firmness rating (E), and mean gonad taste rating (F) for all experimental treatments and wild controls at the end of the experiment. See text for full explanation of gonad quality ratings. Error bars are S.E. and n = 3. Letters above bars indicate the results of a Fisher’s LSD multiple comparisons post-hoc test showing significant pair-wise differences among experimental treatments and wild controls.

320 C.M. Pearce et al. / Aquaculture 214 (2002) 307–332 Fig. 3. Mean percent gonad yield (A), mean gonad colour rating done with paint samples (B), mean gonad colour rating done without paint samples (C), mean gonad texture rating (D), mean gonad firmness rating (E), and mean gonad taste rating (F) for prepared diet treatments only at the end of the experiment. See text for full explanation of gonad quality ratings. Error bars are S.E. and n = 3. Letters above bars or next to % protein legend indicate the results of a Fisher’s LSD multiple comparisons post-hoc test showing significant pair-wise differences among protein source ratios or protein concentrations, respectively.

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

321

Table 3 ANOVA results (repeated four-way) on percent gonad yield, gonad colour, and percent gonad water throughout the 12-week experiment Data set/analysis

SS

df

Gonad Yield/four-way ANOVA Between subjects Source ratio (S) Concentration (C) S
C Block Error Within subjects Time (T) T
S T
C T
S
C T
Block Error

Source

209.22 30.59 55.95 66.52 196.02 7027.70 89.97 37.49 80.42 42.34 323.28

3 2 6 2 22 9 27 18 54 18 198

Gonad Colour/four-way ANOVA Between subjects Source ratio (S) Concentration (C) S
C Block Error Within subjects Time (T) T
S T
C T
S
C T
Block Error

0.30 0.43 0.44 0.12 1.91 2.53 2.23 1.92 3.83 2.57 15.74

3 2 6 2 22 9 27 18 54 18 198

Gonad Water/four-way ANOVA Between subjects Source ratio (S) Concentration (C) S
C Block Error Within subjects Time (T) T
S T
C T
S
C T
Block Error

35.54 74.16 8.13 34.74 62.14 117.32 16.13 14.42 37.42 21.78 113.95

3 2 6 2 22 9 27 18 54 18 198

F-ratio

P-value

7.83 1.72 1.05 3.73

< 0.001**** > 0.1 NS > 0.1 NS < 0.05*

478.25 2.04 1.28 0.91 1.44

< 0.001**** < 0.005*** > 0.1 NS > 0.5 NS > 0.1 NS

1.14 2.51 0.85 0.71

> 0.1 > 0.1 > 0.5 > 0.5

NS NS NS NS

3.54 1.04 1.34 0.89 1.79

< 0.001**** > 0.1 NS > 0.1 NS > 0.5 NS < 0.05*

4.19 13.13 0.48 6.15

< 0.05* < 0.001**** > 0.5 NS < 0.01**

22.65 1.04 1.39 1.20 2.10

< 0.001**** > 0.1 NS > 0.1 NS > 0.1 NS < 0.01**

protein concentration or protein source ratio (Table 3). Two-way interactions between time and protein concentration and time and protein source ratio were non-significant as was the three-way interaction between time, protein concentration, and protein source ratio (Table 3). Gonad colour improved over the experimental period as can be seen by the general decrease in colour ratings over time (Fig. 6). The significant difference between the mean colour ratings done with paint samples for week 12 (2.3 F 0.1) and week 1 (2.5 F 0.1) in the four-way ANOVA (Fig. 6) and the non-significant difference among all

322

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

Fig. 4. Mean percent gonad yield for the four protein source ratios at each sampling period. Error bars are S.E. and n = 9. Letters above bars indicate the results of a Fisher’s LSD multiple comparisons post-hoc test showing significant pair-wise differences among protein source ratios at each sampling period.

treatments (including wild 0-week) in the two-way ANOVA (Fig. 2B), indicates that colour rating in the original sample of 30 individuals at week 0 (2.1 F 0.1) may have been over-estimated (compare this rating with those of weeks 1 – 12 where colour rating was always 2.3 or higher). 3.5. Gonad texture Mean gonad texture ratings for prepared diets at the end of the experiment fell between 1.8 F 0.2 (30:24) and 2.2 F 0.1 (15:19, 30:19, 45:24) (distinct gonad segment halves and smooth texture) (Fig. 2D). Prepared diet treatments did not differ significantly from kelp (1.8 F 0.0) or wild 12-week (2.3 F 0.1) controls (Table 2, Fig. 2D). A three-way ANOVA, examining the effects of protein concentration and protein source ratio on gonad texture ratings at the end of the experiment, showed no significant main effects or interactions (Table 2, Fig. 3D). 3.6. Gonad firmness Mean gonad firmness ratings for prepared diets at the end of the experiment fell between 1.6 F 0.2 (30:24) and 2.2 F 0.4 (0:29) (between very firm and firm) (Fig. 2E). While kelp (1.5 F 0.0) and wild 12-week (1.3 F 0.2) controls had slightly firmer gonads than any of the prepared diets, there was no significant difference among any of the treatments (Table 2, Fig. 2E). A three-way ANOVA, examining the effects of protein concentration and protein source ratio on gonad firmness ratings at the end of the experiment, showed no significant main effects or interactions (Table 2, Fig. 3E).

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

Fig. 5. Mean gonad colour rating done with paint samples in the various prepared diet treatments at each sampling period. Error bars are S.E. and n = 3. 323

324

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

Fig. 6. Mean gonad colour rating done with paint samples at each sampling period (using prepared diet data only). Error bars are S.E. and n = 36. Letters above bars indicate the results of a Fisher’s LSD multiple comparisons posthoc test showing significant pair-wise differences among sampling periods.

3.7. Gonad taste Mean gonad taste ratings for prepared diets at the end of the experiment fell between 3.3 F 0.2 (30:19) and 4.6 F 0.5 (30:29) (most treatments rating between good and satisfactory) (Fig. 2F). A two-way ANOVA conducted on all experimental treatments as well as the wild 12-week sample gave a significant P-value (Table 2). Kelp (2.1 F 0.3) produced significantly better tasting gonads than any prepared diet treatment, while the wild 12-week treatment (2.4 F 0.4) produced better tasting gonads than any prepared diet except 30:19 and 45:19 (3.6 F 0.2) (Fig. 2F). Among the prepared diets, there was only one significant pair-wise comparison: 30:19 had better tasting gonads than 30:29 (Fig. 2F). A three-way ANOVA, examining the effects of protein concentration and protein source ratio on gonad taste ratings at the end of the experiment, showed no significant main effects or interactions (Table 2, Fig. 3F). Although not significant at P = 0.05, there was a slight tendency for taste to get worse with increasing protein concentration ( P = 0.124, Fig. 3F). 3.8. Gonad water Percent gonad water in all prepared diet treatments at each sampling period is shown in Fig. 7. A four-way repeated ANOVA, examining the effects of protein concentration and protein source ratio on percent gonad water over time, revealed significant effects of

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

Fig. 7. Mean percent gonad water in the various prepared diet treatments at each sampling period. Error bars are S.E. and n = 3. 325

326

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

source ratio, concentration, and time (Table 3). All two-way and three-way interaction terms were non-significant (Table 3). For protein concentration averaged across all protein source ratios and sampling periods, the 19% protein treatment (79.0 F 0.1%) had significantly lower percent gonad water than the 24% protein treatment (79.6 F 0.1%) which, in turn, had significantly lower percent gonad water than the 29% treatment (80.1 F 0.1%) (Fig. 8A). For protein source ratio averaged across all protein concentrations and sampling periods, the 45:0:0:55:0 treatment had significantly lower percent gonad water than the three other source ratio treatments, among which there were no significant differences (Fig. 8B). Percent gonad water averaged across all protein source ratios and concentrations gradually declined over the course of the experiment from 80.3 F 0.2% in week 1 to 79.2 F 0.1% in week 12 (Fig. 8C).

Fig. 8. Mean percent gonad water of the various protein concentrations (A), protein source ratios (B), and sampling periods (C). Error bars are S.E. and n = 120, n = 90, n = 36, respectively. Letters above bars indicate the results of a Fisher’s LSD multiple comparisons post-hoc test showing significant pair-wise differences among protein concentrations (A), protein source ratios (B), or sampling periods (C).

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

327

4. Discussion 4.1. Gonad yield Percent gonad yield of urchins fed prepared diets increased significantly more than that of individuals fed kelp or those sampled from the wild. Percent yield increases per week reached a maximum of 1.3% for prepared diets (i.e. 15:29 treatment), but was only 0.5% for the kelp control. Similar high rates of gonad yield increases for S. droebachiensis fed prepared diets have been previously reported (Klinger et al., 1997: 1.4% week 1; Motnikar et al., 1997: 1.2 –2.6% week 1; Ha´vardsson et al., 1999: 0.7– 0.8% week 1; Pearce et al., 2002: 1.2– 1.4% week 1; Robinson et al., in press: 1.6 –2.2% week 1; see review table in Robinson et al., in press). There is little doubt then that prepared diets can be successfully utilized to increase the gonad yield of S. droebachiensis under culture conditions. Further developments are required to refine diets to optimize both gonad colour and taste (see Gonad colour and Gonad taste). de Jong-Westman et al. (1995a) previously reported significantly higher percent gonad yields in adults of S. droebachiensis (test diameter range: 50 –70 mm) fed prepared diets containing 20% protein (i.e. ground wheat, condensed fish solubles, and albumin) than in those given similar diets with only 10% protein. Akiyama et al. (2001) fed young P. depressus (mean test diameter: 15 mm) prepared diets containing 10%, 20%, 30%, 40%, or 50% crude protein (i.e. casein) for a period of 56 days. They found that urchins fed the 20%, 30%, or 40% protein diets had higher gonad indices (7.15%, 7.27%, and 6.76%, respectively) at the end of the experiment than those individuals given the 10% or 50% protein diets (5.81% and 5.62%, respectively), but noted that these differences were not statistically significant. In the present study, no significant difference was found in percent gonad yields of urchins given prepared diets with 19%, 24%, or 29% protein, but all prepared diets produced significantly greater yields than a control diet of kelp which typically has a substantially lower protein content [Cho et al., 1995 reported that L. japonica had a crude protein concentration of 8.7% (percentage composition by dry weight averaged over 4– 5 months)]. Taken together, the results of these three studies suggest that sea urchin gonad enhancement is most likely maximized at a dietary protein concentration between 10% and 19%. Higher protein levels are unlikely to significantly increase gonad yield, but may add substantially to the price of diet formulation (depending on the protein source) and/or decrease gonad quality (see Gonad colour and Gonad taste). Interestingly, protein concentration of prepared diets at or above 20% does not significantly alter somatic growth rates of juvenile or adult sea urchins (de Jong-Westman et al., 1995a; McBride et al., 1998; Akiyama et al., 2001; Kennedy et al., 2001). A number of studies have documented the effect—or lack of effect—of varying protein source in prepared diets on gonad yield of adult urchins. Lawrence et al. (1992) fed two size groups of P. lividus [small and large (exact size not given)], prepared diets with either soybean meal protein only or fish meal protein only. They found that, with both sizes of urchins, gonad indices did not differ significantly with diet. Fernandez and Boudouresque (1998) fed small P. lividus (mean test diameter: 23.2 mm) three different prepared diets: ‘‘vegetable based’’ [low protein, high carbohydrate (58%)], ‘‘animal based’’ [high protein (47%), low carbohydrate], and ‘‘mixed based’’ [animal and vegetable matter: intermediate

328

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

protein (29%), intermediate carbohydrate (35%)]. They found that gonad growth was best on the mixed diet. Similarly Fernandez and Boudouresque (2000) fed three size groups of P. lividus (mean test diameters: 23.2, 32.1, 42.5 mm) vegetable-based, animal-based, and mixed diets (protein, carbohydrate levels: 13%, 58%; 47%, 16%; 29%, 35%, respectively). They found that gonad indices for urchins in the two smaller size groups were higher for the mixed diet than for the other two diets while urchins in the largest size group had approximately the same gonad yield with all three diets. Barker et al. (1998) fed three size groups of E. chloroticus (test diameter ranges: 30 – 40, 50– 60, 70 –80 mm) prepared diets with fish meal and soybean meal combined or soybean meal only. Results were complex and dependent on size class and time of year the experiment was run, but in the cases where there was a significant difference between the two diets, gonad indices were greater in the diets with added fish meal. Along with Fernandez and Boudouresque (1998, 2000), we found that highest gonad indices were achieved using diets with intermediate levels of animal protein (i.e. 10% of total protein meal weight attributed to fish meal). Gonad yields were significantly lower in diets without animal protein and in those with the highest level of animal protein (i.e. 15% of total protein meal weight attributable to fish meal). Unlike Fernandez and Boudouresque (1998, 2000) and Barker et al. (1998), the present study had equivalent protein concentrations for each protein source ratio tested, so that the effect on gonad yield is truly that of varying protein source ratio and not differences in protein concentration. This suggests that different protein sources may be utilized differently in providing energy for gonad development. Since the present study utilized a mixture of protein sources, it cannot be determined what specific meal(s) were most important in gonad production. Further research in this area is required to determine differences among actual protein sources. 4.2. Gonad colour Prepared diets produced gonad colours at the end of the experiment that were, generally, acceptable for the commercial roe industry (i.e. pale yellow/orange to yellowbrown/orange-brown) and not significantly different from kelp-fed or wild-caught urchins. Colour of urchin gonads in the present study, using a h-carotene concentration of 0.3 mg kg 1 dry weight of dietary ingredients, was markedly improved from that of a previous study (Pearce et al., 2002) using a similar prepared diet with only 0.2 mg kg 1 of hcarotene; gonad colour in this earlier study was, generally, tan or cream. Production of appropriate gonad colours in sea urchins fed prepared diets can been problematic if appropriate levels of certain dietary pigment sources are not included in the diet (Barker et al., 1998; Grosjean et al., 1998; Watts et al., 1998; Pearce et al., 2002). Recent research by Robinson et al. (in press) and Pearce (unpublished observations) with S. droebachiensis has shown that h-carotene is a very effective pigment source for producing the bright yellow/orange colouration sought after by the commercial market, but that the most effective concentration is f 200 –250 mg kg 1, substantially higher than that used in the current study. Robinson et al. (in press) have also suggested that pigment origin can affect colour production. They tested two sources of h-carotene: (1) natural, spray-dried preparation of the phytoplankton Duneliella salina and (2) synthetically produced by chemical means

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

329

and sold as an animal feed supplement. They found that h-carotene derived from natural phytoplankton produced better gonad colour than that derived synthetically and attributed this to differences in the form of h-carotene of the two sources (i.e. mixture of cis and trans isomers for the former and all trans for the latter) and/or differences in other components included with the h-carotene products (i.e. algal cellular components and other pigments such as lutein in the former and wheat middlings in the latter). Interestingly, the algal-derived h-carotene produced better tasting gonads than the synthetic product (Pearce, unpublished observations). In the colour rating with paint samples data set, gonad colour was affected by protein concentration, the 19% and 24% levels producing significantly better colour than the 29% level. This trend was, generally, present in the colour rating without paint samples data as well, but there was no significant difference among the protein concentrations. How protein level may affect gonad colour is not entirely obvious since carotenoid pigments such as h-carotene and echinenone are thought to be the primary pigment molecules in gonads of S. droebachiensis and other echinoids (see review by Matsuno and Tsushima, 2001). In the urchin Lytechinus variegatus, higher protein levels led to decreases in carbohydrate (mostly glycogen) concentration in the gonads (S.A. Watts, University of Alabama, personal communication) and decreasing gonad glycogen may lead to a darkening of gonad colour. Pearce et al. (2002), working with S. droebachiensis, found that prepared diets bound with corn starch produced significantly better gonad colour than prepared diets formulated with other binders such as gelatin, guar gum, or sodium alginate and hypothesized that starch may have led to greater production of storage glycogen and, thus, a lighter or whiter gonad background and a brighter gonad colour. An alternate, but not mutually exclusive hypothesis, is that the development of suitable gonad colour may be affected by certain competing metabolic pathways at higher protein levels. Further research is required in this area, but it should be noted that production of desirable gonad colours in sea urchins fed prepared diets may not be a simple case of the addition of appropriate levels of required pigmentation. Other dietary components and interacting metabolic pathways must also be taken into consideration. 4.3. Gonad texture and firmness Gonads from urchins fed prepared diets had very good texture (i.e. smooth to very smooth with distinct gonad segment halves) and firmness (i.e. firm to very firm) at the end of the experiment. While kelp-fed individuals had slightly better gonad texture and firmness than those fed prepared diets, the differences were slight and not significant. Prepared diets produced highly marketable gonads in terms of both texture and firmness. Interestingly, although protein concentration did not significantly affect gonad firmness nor percent gonad yield, the amount of water in the gonads during the experiment increased significantly with increasing protein level. This trend of elevated gonad water with increasing protein concentration has also been found in L. variegatus and is hypothesized to be connected with the hydration state of macro-nutrients stored in the gonads (S.A. Watts, University of Alabama, personal communication). While statistically significant, the actual differences in the amount of gonad water among the protein concentrations in the present study was small (79.0% for 19% protein, 79.6% for 24%

330

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

protein, 80.1% for 29% protein) and statistical significance was due to extremely small variation within treatments. The biological and/or commercial importance of this statistical dissimilarity is unlikely to be highly relevant. Gonad firmness did decrease slightly with increasing protein concentration, but not significantly and only in two of the four meal source ratios (i.e. 0:30:30:25:15 and 45:0:0:55:0). This indicates that differences in gonad water did not significantly impact gonad firmness. The amount of water in the gonads was also significantly affected by protein source ratio. Again, the actual differences among the treatments were small and the biological and/or commercial importance of this statistical finding is questionable. 4.4. Gonad taste Gonad taste is extremely important in the commercial roe industry, but very few enhancement studies using prepared or natural diets have examined this quality. Gonads from urchins fed prepared diets in this study varied substantially from bitter to sweet but, generally, tasted significantly worse than kelp-fed or wild-caught individuals, the taste of which was typically very sweet. Since fish meal and/or fish oil products have been previously associated with poor gonad flavour (Hoshikawa et al., 1998; Pearce et al., 2002), it was hypothesized prior to experimentation that increasing the contribution of fish meal in the diet from 0% to 15% of the total protein meal weight would decrease taste acceptability. Gonad taste was not significantly dependent on protein source ratio, however, indicating that fish meal—at least at the levels used in this study—did not negatively impact flavour. Gonad taste was not significantly affected by protein concentration, but there was a slight trend of worsening flavour with increasing protein level. Amino acid profiles of gonads from Japanese wild urchins (Heliocidaris crassispina, S. intermedius, S. nudus, S. pulcherrimus) suggest that good flavour may be associated with high levels of certain amino acids such as alanine, arginine, glutamic acid, glycine, lysine, serine, and/or taurine (Komata et al., 1962; Hirano et al., 1978) while bitter flavour is associated with high concentrations of valine and pulcherrimine (Hoshikawa et al., 1998; Murata et al., 2001). While amino acid profiles of the resultant gonads were not characterized in the present study, higher protein concentrations may have lead to an accumulation of valine and/or pulcherrimine. Further dietary research, including amino acid profiling, is required to optimize gonad taste of urchins fed prepared diets.

Acknowledgements The National Research Council of Canada Industrial Research Assistance Program, the Atlantic Canada Opportunities Agency, and Ross Island Salmon (RIS) provided project funding. CMP was partially supported by an Industrial Research Fellowship from the Natural Sciences and Engineering Research Council of Canada. Many thanks are expressed to Ken Brown (RIS president) who had the foresight and fortitude to help initiate and fund this experimental work. We thank Annise Brown and Blaine Brown, the technicians who worked on this project and spent countless hours cracking open sea

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

331

urchins. We are also indebted to Robert Young for helping in the collection of sea urchins and to Wade Blanchard (Statistical Consulting Service of Dalhousie University) for statistical advice.

References Akiyama, T., Unuma, T., Yamamoto, T., Furuita, H., Konishi, K., 1997. An evaluation of amino acid sources and binders in semipurified diet for red sea urchin Pseudocentrotus depressus. Fish. Sci. 63, 881 – 886. Akiyama, T., Unuma, T., Yamamoto, T., 2001. Optimum protein level in a purified diet for young red sea urchin Pseudocentrotus depressus. Fish. Sci. 67, 361 – 363. Barker, M.F., Keogh, J.A., Lawrence, J.M., Lawrence, A.L., 1998. Feeding rate, absorption efficiencies, growth, and enhancement of gonad production in the New Zealand sea urchin Evechinus chloroticus Valenciennes (Echinoidea: Echinometridae) fed prepared and natural diets. J. Shellfish Res. 17, 1583 – 1590. Cho, D.-M., Kim, D.-S., Lee, D.-S., Kim, H.-R., Pyeun, J.-H., 1995. Trace components and functional saccharides in seaweed. 1. Changes in proximate composition and trace elements according to harvest season and places. Bull. Korean Fish. Soc. 28, 49 – 59. de Jong-Westman, M., March, B.E., Carefoot, T.H., 1995a. The effect of different nutrient formulations in artificial diets on gonad growth in the sea urchin Strongylocentrotus droebachiensis. Can. J. Zool. 73, 1495 – 1502. de Jong-Westman, M., Qian, P.-Y., March, B.E., Carefoot, T.H., 1995b. Artificial diets in sea urchin culture: effects of dietary protein level and other additives on egg quality, larval morphometrics, and larval survival in the green sea urchin, Strongylocentrotus droebachiensis. Can. J. Zool. 73, 2080 – 2090. Fernandez, C., 1997. Effect of diet on the biochemical composition of Paracentrotus lividus (Echinodermata: Echinoidea) under natural and rearing conditions (effect of diet on biochemical composition of urchins). Comp. Biochem. Physiol. 118A, 1377 – 1384. Fernandez, C., Boudouresque, C.-F., 1998. Evaluating artificial diets for small Paracentrotus lividus (Echinodermata: Echinoidea). In: Mooi, R., Telford, M. (Eds.), Echinoderms: San Francisco. Balkema, Rotterdam, pp. 651 – 656. Fernandez, C., Boudouresque, C.-F., 2000. Nutrition of the sea urchin Paracentrotus lividus (Echinodermata: Echinoidea) fed different artificial food. Mar. Ecol.: Prog. Ser. 204, 131 – 141. Fernandez, C., Caltagirone, A., 1994. Growth rate of adult sea urchins, Paracentrotus lividus in a lagoon environment: the effect of different diet types. In: David, B., Guille, A., Fe´ral, J.-P., Roux, M. (Eds.), Echinoderms Through Time. Balkema, Rotterdam, pp. 655 – 660. Fernandez, C., Pergent, G., 1998. Effect of different formulated diets and rearing conditions on growth parameters in the sea urchin Paracentrotus lividus. J. Shellfish Res. 17, 1571 – 1581. Goebel, N., Barker, M.F., 1998. Artificial diets supplemented with carotenoid pigments as feeds for sea urchins. In: Mooi, R., Telford, M. (Eds.), Echinoderms: San Francisco. Balkema, Rotterdam, pp. 667 – 672. Grosjean, P., Spirlet, C., Gosselin, P., Vaı¨tilingon, D., Jangoux, M., 1998. Land-based, closed-cycle echiniculture of Paracentrotus lividus (Lamarck) (Echinoidea: Echinodermata): a long-term experiment at a pilot scale. J. Shellfish Res. 17, 1523 – 1531. Ha´vardsson, B., Imsland, A.K., Christiansen, R., 1999. The effect of astaxanthin in feed and environmental temperature on carotenoid concentration in the gonads of the green sea urchin Strongylocentrotus droebachiensis Mu¨ller. J. World Aquacult. Soc. 30, 208 – 218. Hirano, T., Yamazawa, S., Suyama, M., 1978. Chemical composition of gonad extract of sea-urchin, Strongylocentrotus nudus. Bull. Jpn. Soc. Sci. Fish. 44, 1037 – 1040 (in Japanese, with English abstract). Hoshikawa, H., Takahashi, K., Sugimoto, T., Tuji, K., Nobuta, S., 1998. The effects of fish meal feeding on the gonad quality of cultivated sea urchins, Strongylocentrotus nudus (A. Agassiz). Sci. Rep. Hokkaido Fish. Exp. Stn. 52, 17 – 24 (in Japanese, with English abstract). Keesing, J.K., Hall, K.C., 1998. Review of harvests and status of world sea urchin fisheries points to opportunities for aquaculture. J. Shellfish Res. 17, 1597 – 1604. Kennedy, E.J., Robinson, S.M.C., Parsons, G.J., Castell, J., 2001. Studies on feed formulations to maximize

332

C.M. Pearce et al. / Aquaculture 214 (2002) 307–332

somatic growth rates of juvenile green sea urchins (Strongylocentrotus droebachiensis). Aquacult. Assoc. Can. Spec. Publ. 4, 68 – 71. Klinger, T.S., Lawrence, J.M., Lawrence, A.L., 1994. Digestive characteristics of the sea-urchin Lytechinus variegatus (Lamarck) (Echinodermata: Echinoidea) fed prepared feeds. J. World Aquacult. Soc. 25, 489 – 496. Klinger, T.S., Lawrence, J.M., Lawrence, A.L., 1997. Gonad and somatic production of Strongylocentrotus droebachiensis fed manufactured feeds. Bull. Aquacult. Assoc. Can. 97 (1), 35 – 37. Komata, Y., Kosugi, N., Ito, T., 1962. Studies on the extractives of ‘‘uni’’: I. Free amino acid composition. Bull. Jpn. Soc. Sci. Fish. 28, 623 – 629 (in Japanese, with English abstract). Lares, M.T., McClintock, J.B., 1991. The effects of food quality and temperature on the nutrition of the carnivorous sea urchin Eucidaris tribuloides (Lamarck). J. Exp. Mar. Biol. Ecol. 149, 279 – 286. Lawrence, J., Fenaux, L., Corre, M.C., Lawrence, A., 1992. The effect of quantity and quality of prepared diets on production in Paracentrotus lividus (Echinodermata: Echinoidea). In: Scalera-Liaci, L., Canicattı`, C. (Eds.), Echinoderm Research 1991. Balkema, Rotterdam, pp. 107 – 110. Lawrence, J.M., Olave, S., Otaiza, R., Lawrence, A.L., Bustos, E., 1997. Enhancement of gonad production in the sea urchin Loxechinus albus in Chile fed extruded feeds. J. World Aquacult. Soc. 28, 91 – 96. Levin, V.S., Naidenko, V.P., 1987. Artificial diet for laboratory-maintained sea urchin Strongylocentrotus intermedius. Sov. J. Mar. Biol. 13, 344 – 349. Matsuno, T., Tsushima, M., 2001. Carotenoids in sea urchins. In: Lawrence, J.M. (Ed.), Edible Sea Urchins: Biology and Ecology. Elsevier, New York, pp. 115 – 138. McBride, S.C., Pinnix, W.D., Lawrence, J.M., Lawrence, A.L., Mulligan, T.M., 1997. The effect of temperature on production of gonads by the sea urchin Strongylocentrotus franciscanus fed natural and prepared diets. J. World Aquacult. Soc. 28, 357 – 365. McBride, S.C., Lawrence, J.M., Lawrence, A.L., Mulligan, T.J., 1998. The effect of protein concentration in prepared feeds on growth, feeding rate, total organic absorption, and gross assimilation efficiency of the sea urchin Strongylocentrotus franciscanus. J. Shellfish Res. 17, 1563 – 1570. McBride, S.C., Lawrence, J.M., Lawrence, A.L., Mulligan, T.J., 1999. Ingestion, absorption, and gonad production of adult Strongylocentrotus franciscanus fed different rations of a prepared diet. J. World Aquacult. Soc. 30, 364 – 370. Motnikar, S., Bryl, P., Boyer, J., 1997. Conditioning green sea urchins in tanks: the effect of semi-moist diets on gonad quality. Bull. Aquacult. Assoc. Can. 97 (1), 21 – 25. Murata, Y., Sata, N.U., Yokoyama, M., Kuwahara, R., Kaneniwa, M., Oohara, I., 2001. Determination of a novel bitter amino acid, pulcherrimine, in the gonad of the green sea urchin Hemicentrotus pulcherrimus. Fish. Sci. 67, 341 – 345. Olave, S., Bustos, E., Lawrence, J.M., Ca´rcamo, P., 2001. The effect of size and diet on gonad production by the Chilean sea urchin Loxechinus albus. J. World Aquacult. Soc. 32, 210 – 214. Pearce, C.M., Daggett, T.L., Robinson, S.M.C., 2002. Effect of binder type and concentration on prepared feed stability and gonad yield and quality of the green sea urchin, Strongylocentrotus droebachiensis. Aquaculture 205, 301 – 323. Robinson, S.M.C., Castell, J.D., Kennedy, E.J., 2002. Developing suitable colour in the gonads of cultured green sea urchins (Strongylocentrotus droebachiensis). Aquaculture (in press). Spirlet, C., Grosjean, P., Jangoux, M., 2000. Optimization of gonad growth by manipulation of temperature and photoperiod in cultivated sea urchins, Paracentrotus lividus (Lamarck) (Echinodermata). Aquaculture 185, 85 – 99. Unuma, T., Yamamoto, T., Akiyama, T., 1999. Effect of steroids on gonadal growth and gametogenesis in the juvenile red sea urchin Pseudocentrotus depressus. Biol. Bull. 196, 199 – 204. Walker, C.W., Lesser, M.P., 1998. Manipulation of food and photoperiod promotes out-of-season gametogenesis in the green sea urchin, Strongylocentrotus droebachiensis: implications for aquaculture. Mar. Biol. 132, 663 – 676. Watts, S.A., Boettger, S.A., McClintock, J.B., Lawrence, J.M., 1998. Gonad production in the sea urchin Lytechinus variegatus (Lamarck) fed prepared diets. J. Shellfish Res. 17, 1591 – 1595.