Gamete maturation and gonad growth in fed and starved sea urchin paracentrotus lividus (Lamarck, 1816).
Article Type: Report
Subject: Germ cells (Research)
Gonads (Research)
Gonads (Growth)
Sea urchins (Research)
Sea urchins (Physiological aspects)
Sea urchins (Food and nutrition)
Animal feeding and feeds (Research)
Authors: Fabbrocini, Adele
D'Adamo, Raffaele
Pub Date: 12/01/2010
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2010 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: Dec, 2010 Source Volume: 29 Source Issue: 4
Topic: Event Code: 310 Science & research Computer Subject: Company growth
Product: Product Code: 2048000 Prepared Feeds; 3523860 Livestock Feeders NAICS Code: 311119 Other Animal Food Manufacturing; 333111 Farm Machinery and Equipment Manufacturing
Geographic: Geographic Scope: Italy Geographic Code: 4EUIT Italy
Accession Number: 247523226
Full Text: ABSTRACT A 4-wk rearing trial of the sea urchin Paracentrotus lividus was carried out in a closed-circuit system in the presence and absence of food supply to evaluate the short-term response of gametogenesis to different feeding conditions. At the end of the trial, the gonad index (GI) was calculated, histological analyses of the gonads were performed, gamete fertilization ability was evaluated, and sperm motility was assessed by computerized motility analysis. Starvation significantly affected gametogenesis, whereas developing gametes were always observed in fed animals, whose GI had doubled by the end of the 4-wk trial. No differences were recorded between gametes from reared (fed) urchins and wild-collected ones. Although spent gonads frequently contained unspawned motile spermatozoa or morphologically intact eggs, the fertilization ability of gametes from starved urchins was significantly lower. It may be concluded that, although they were at the end of the reproductive season, the specimens fed ad libitum were able to reactivate the gamete maturation process immediately. This ability can be used in short-term procedures for roe enhancement and gamete production, particularly for specimens from areas in which environmental conditions determine slow gametogenesis and a consequently short reproductive season.

KEY WORDS: sea urchin, Paraeentrotus lividus, gonad index, gonad histology, computerized sperm motility analysis, fertilization

INTRODUCTION

The regular echinoid Paracentrotus lividus (Lamarck, 1816) is a macroalgivore sea urchin commonly found along the North Atlantic coasts from Ireland to southern Morocco and throughout the Mediterranean Sea (Bayed et al. 2005, Symonds et al. 2007). The P. lividus gonads, commonly called "roe," are a luxury food particularly appreciated in many parts of Europe, especially in France and southern Italy. Because wild stocks of sea urchins are declining in European coastal waters, mainly as a result of habitat change and overfishing (Sala & Zabala 1996, Lesser & Walker 1998, Guidetti et al. 2004), during the past decade there has been a significant increase in efforts to develop cost-effective ways of culturing sea urchins. Research into the farming of sea urchins has followed two different objectives: the short-time rearing of wildcaught specimens to increase out-of-season gonadal yield (Russell 1998, Pearce et al. 2004, Shpigel et al. 2005), and the regulation of the reproductive cycle in controlled conditions for the long-term production of gametes and embryos to be used for generating larvae and thus new stocks (Grosjean et al. 1998, Daggett et al. 2005).

Moreover, sea urchins are commonly used as a model in developmental biology research (Berdyshev et al. 1995, Kominami & Takata 2004, Kaupp et al. 2006) and as a test species in ecotoxicological bioassays (Pagano et al. 1993, Fabbrocini et al. 2005, Marin et al. 2007). There is, therefore, a need for continuous production of viable sea urchin gametes and embryos regardless of the seasonal reproductive cycle (Schipper et al. 2008, Paredes & Bellas 2009). Such production needs to be carried out in accordance with standardized environmental parameters and feeding protocols to guarantee constant and homogeneous gamete quality, and in laboratory-scaled closed rearing systems that can be easily managed even at some distance from the sea (Leahy et al. 1981, Grosjean et al. 1998).

Sea urchin biology in general has been well studied, and much research has been carried out to determine all the phases of the reproductive cycle in relation to environmental characteristics (Byrne 1990, Lozano et al. 1995, Spirlet et al. 1998, Sanchez-Espana et al. 2004, Barbaglio et al. 2007). Much research has also been carried out into the induction of alterations to the gametogenic cycle while rearing the animals in confinement, by manipulating food availability, temperature, and photoperiod (Hiratsuka & Uehara 2007, Siikavuopio et al. 2007, Siikavuopio et al. 2008, Gibbs et al. 2009, Phillips et al. 2009), which are important triggers of the gametogenic cycle in both P. lividus and other sea urchin species in natural conditions (Byrne 1990, Spirlet et al. 1998). Because most research is aimed at the production of large gonads to be consumed as food, a frequently used parameter for evaluating gonad growth is the gonad index (GI = gonad wet weight (measured in grams) / urchin wet weight (measured in grams) x 100). On the other hand, both storage of nutrients in nutritive phagocytes, which characterizes the early maturation stages (Byrne 1990), and the presence of large amounts of mature gametes (late maturation stages) can be responsible for increased gonad size. Because GI does not distinguish between them, it cannot, therefore, give information on the gametogenic stage (Spirlet et al. 1998, Walker & Lesser 1998, Garrido & Barber 2001, Barbaglio et al. 2007). In addition, most studies have focused on the effects of medium- to long-term rearing periods (6-24 wk (Spirlet et al. 2000, Shpigel et al. 2005, Cook & Kelly 2007)).

Given that sea urchin gonads are capable of responding to abrupt variations in the availability and quality of food even within the space of a few days (Russell 1998), a histological evaluation of the changes occurring in the gonads during the early phases of rearing in controlled conditions is of crucial importance to set up rearing protocols for short-term induction of gamete maturation that are specifically tailored to the different initial gonadal conditions of reared sea urchin specimens.

The aim of this research is to evaluate the short-term response of P. lividus gonads to the presence or absence of food supply during a 4-wk rearing trial in a closed-circuit system, as a part of a wider research project focusing on sea urchin gonad conditioning for the continuous production of gametes and embryos to be used in marine biology research and in ecotoxicological tests. Gonad yield, histological evaluation of gonad stage, and gamete quality assessment are used as evaluation parameters to obtain information about changes to both gonad tissue and gamete viability that occur when sea urchin are reared for short periods in small-volume recirculating aquaria.

MATERIALS AND METHODS

Sea Urchin Collection

Sea urchins (P. lividus) were hand collected with the aid of scuba-diving equipment in May 2005 and June 2005 from an artificial rock ledge near Termoli (41[degrees]54'N, 16[degrees]10'E) on the southern Adriatic coast of Italy, placed in a cooler, and carried to the laboratory under moist conditions within 2 h.

Experimental Design

On their arrival in the laboratory, the sea urchins were measured (horizontal diameter) by caliper (0.05-min accuracy), and adult specimens (with a test diameter of between 35 mm and 45 mm, excluding spines) were selected for the experimental trials. This class size was chosen because the level of energy used for reproduction is higher than it is for juveniles, which in contrast allocate more energy to test and lantern growth (Fernandez & Boudouresque 2000).

The selected sea urchins were placed in a recirculating aquarium and acclimatized for 5 days to the confined rearing conditions. The closed rearing system consisted of 6 rectangular interconnected tanks (100 L each) with an external filter, denitrator, protein skimmer, ultraviolet sterilizer, refrigerator, and centrifugal pump that recirculated natural seawater (from the Tremiti islands Marine Protected Area, southern Adriatic Sea) with a flow of 50 L/min. Aeration in the tanks provided additional water movement and air supply to the urchins. The abiotic parameters were measured daily by multiparametric probe (YS16920, Hertfordshire, UK) and kept at constant temperature (18 [+ or -] 1[degrees]C), salinity (36 [+ or -] 1 psu), pH (8.00-8.20), and dissolved oxygen (>90%). Temperature and salinity ranges reflected field conditions, as recorded on sampling. Because the rearing system was adjacent to the windows, the entire experiment was kept in a natural photoperiod (15 h light/9 h darkness). The stock density was 5 L per individual. These rearing conditions were found to be optimal in a preliminary 6-wk P. lividus rearing trial (Di Matteo et al. 2006). Plastic mesh was used in the tanks to prevent cross-migration of sea urchins assigned to different feeding regimes.

At the end of the 5-day acclimatization period, injured individuals were discarded and 30 randomly selected specimens were sacrificed to determine the GI, perform histological analyses, and evaluate gamete quality (t0 values).

The remaining 120 sea urchins were randomly divided into 2 experimental groups (60 for each group): the first group of animals were denied food, whereas the second were fed ad libiturn on pelletized (2.5 x 2.5 x5 mm) formulated feed (Classic K; hendrix SpA, Mozzecane-VR, Italy) for up to 4 wk. Twice a week the tanks were cleaned and uneaten feed was removed and replaced; feed was supplied at a rate of 2% of sea urchin biomass per day.

The formulated feed contained soybean meal, wheat meal, fish meal, sunflower meal, fish oil, soybean oil, rapeseed oil, dicalcium phosphate, vitamin A, vitamin D3, vitamin E, and BHT. Its approximate nutritional values are shown in Table 1.

At the end of the 4 wk, 30 sea urchins were again collected in the same site as a control for field conditions (Field, 4 wk).

Evaluation Parameters

After 2 wk of rearing, 30 urchins from each experimental group were sacrificed to perform histological analyses. At the end of the rearing period (4 wk), the remaining 30 urchins from each experimental group were sacrificed to determine the GI, perform histological analysis, and evaluate gamete quality. All these parameters were also determined in the urchins collected in the field at t0 and at the end of the rearing trial.

Gonad Index

Individuals were allowed to drip for approximately 5 min, weighed (0.2-mg accuracy) and then dissected. The gonads were extracted and fresh weighed for the following GI evaluation:

GI = gonad wet weight (in grams)/sea urchin wet weight(in grams) x 100.

Histological Evaluation

One of the 5 gonads of each animal was fixed in 10% formalin and embedded in paraffin; 7 [micro]m sections were obtained by microtome and placed on slides. Ten slides for each gonad were randomly selected and stained with Mayer's haemalum and eosin (5 slides) and with Masson trichrome (5 slides). The slides were observed by microscope and each was assigned a reproductive stage using the nomenclature in Byrne (1990): stage I, recovery; stage II, growing; stage III, premature; stage IV, mature; stage V, spawning; stage VI, spent.

Gamete Evaluation

Each individual was sexed by examining the extruded gametes. An aliquot of sperm or eggs from each animal was diluted in artificial seawater (ASW, 35 [per thousand] (American Society for Testing and Materials 1998) and observed under a microscope for a preliminary evaluation of sperm motility and egg morphology. The percentage of individuals with visually assessed good gamete quality was calculated, and pools of the best males and females were created for the evaluation of fertilization ability. Briefly, dry sperm was diluted in ASW at a ratio of 1:1,000, added to ASW-washed eggs at a sperm-to-egg ratio of 15,000:1, and incubated in 10 mL polystyrene multiwell dishes until plutei larvae were obtained (72 h, 18[degrees]C, in the dark). Samples were preserved in concentrated buffered formalin, and the percentage of plutei with normal development was scored by observing 200 larvae for each sample (Pagano et al. 1993). Six replicates for each experimental group were performed.

Sperm Motility Analysis

For the male sea urchins reared for 4 wk and the specimens collected in the field at t0 and at the end of the rearing trial (4-wk field), computer-assisted sperm motility analyses were also performed. Dry sperm were diluted in ASW at a ratio of 1:1,000. Sperm movement was recorded using a 100 frame/sec camera (Basler, 782 x 582 resolution) attached to a microscope (Nikon Eclipse E600) with a phase-contrast objective (10 x 10 magnification) connected to a computerized motion analysis system (the Sperm Class Analyzer (SCA), Microptic, S.L., Spain). The SCA acquisition parameters were set as follows: maximum area, 400 [[micro]m.sup.2]; minimum area, 50 [[micro]m.sup.2]; frame rate, 100/sec; total captured images, 100.

For each semen sample, 6 motility records were taken in 6 different microscopic fields; each record consisted of the mean of 3 replicates, each analyzing from 250-500 sperm tracks. The following motion parameters were assessed:

1. Sperm motility classes: Percentages of rapid (velocity > 100 [micro]m/sec), medium (45 [micro]m/sec < velocity < 100 [micro]m/sec), slow (10 [micro]m/sec < velocity < 45 [micro]m/sec), and static (velocity < 10 [micro]m/sec) spermatozoa

2. Curvilinear velocity (VCL, measured in micrometers per second): the velocity of the sperm head along its real curvilinear track, as perceived in 2 dimensions using the microscope

3. Straight-line velocity (VSL, measured in micrometers per second): the velocity of the sperm head along its linear track between its initial and final positions

4. Average path velocity (VAP, measured in micrometers per second): the velocity of the sperm head along its spatial average trajectory

Statistical Analyses

At the end of the rearing trial, the statistical differences in the evaluation parameters among the 3 experimental groups (fed, starved, or wild) were determined (McDonald 2009). The data on GIs did not pass the homogeneity of variance test, so nonparametric statistical analyses were performed. The differences in GI between the males and females of each experimental group were analyzed using the Mann-Whitney U test. Because the differences were not found to be significant, male and female data were combined and the statistical significance of the differences in GIs in the 3 experimental groups was determined using the nonparametric Kruskal-Wallis analysis of variance (ANOVA) on ranks.

The statistical significance of the differences in the percentages of total motile and rapid spermatozoa, and in fertilization success were determined using l-way ANOVA. When significant differences were recorded, the Tukey multiple comparison test was conducted. Prior to analysis, percentage data were arcsine transformed and tested for normality using the Shapiro-Wilk's test, and for homogeneity of variance using Cochran's test. The sperm velocity data (VCL, VSL, and VAP) did not pass the homogeneity of variance test; they were therefore analyzed using the nonparametric Kruskal-Wallis ANOVA on ranks. No quantitative analysis was conducted of histological preparations. All analyses were performed with the StatSoft, Inc. (2008) STATISTICA data analysis software system, version 8.0.

RESULTS

Sea urchin mortality occurred only during the first few days of acclimatization. No sea urchins died during the 4 wk of experimental rearing.

Gonad Index

The GI values for both male and female sea urchins collected in the field (t0 and field, 4 wk) and at the end of the experiment (4 wk rearing) are shown in Table 2.

No differences were recorded using the Mann-Whitney U test in relation to sex for any experimental group (U = 98.50, P = 0.56 for fed sea urchins; U = 102.00, P = 0.66 for starved sea urchins; U = 77.00, P = 0.14 for field-collected sea urchins).

The Kruskal-Wallis test shows that at the end of the rearing trial, GI values were significantly different ([H.sub.2, 90] = 35.437, P < 0.001). The unplanned comparisons of mean ranks show that the GI of fed urchins was significantly higher than that of the starved animals and wild-collected ones (P < 0.001).

Histological Examination

The histological patterns of the gonads are shown in Figures 1 and 2. They were classified into 6 stages according to Byrne (1990). Figure 3 shows the relative frequencies of the recorded gonadal stages at the beginning (t0) and during the 4 wk of trial. Because no substantial differences between the stage frequencies of ovaries and testes were observed, the data were combined for easier discussion of results.

On collection (t0), sea urchins were found to be in different phases of the gonadic cycle. About 20% of individuals were in the growing phase (stage II), with gonads full of developing gametes. Ovary sections (Fig. 1D-F) had growing oocytes surrounded by nutritive phagocytes along the ascinal walls, whereas in the testes (Fig. 2B, C), columns of spermatocytes were migrating toward the center of the ascini, where a meshwork of nutritive phagocytes was present. Similar percentages of animals were in slightly more advanced phases of the gonadic cycle (stages III and IV, 14% and 6%, respectively), with the center of the ascini increasingly filled with mature gametes (Figs. 1G and 2D, E). A small percentage (10%) of the urchins was in the spawning phase (stage V)--both male and female gonads had ascini with spaces vacated by shed gametes, whereas gametogenesis to replace the spawned gametes continued (Figs. 1H and 2F, G). More than 35% of the animals were in the spent stage (stage VI): Unspawned gametes were still present, but no developing gametes were observed along the ascinal wall (Figs. 11 and 2H). Finally, about 15 % of urchins were in the recovery stage (stage I): In the testes, the meshwork of phagocytes was evident (Figs. 2A-I), although in some cases unspawned gametes were still copiously present. In the female gonads, ova in the process of lysis were sometimes present (Fig. 1B), whereas nutritive phagocytes were forming a meshwork across the ascini. In a more advanced recovery phase, a thin layer of developing oocytes was observed (Fig. 1C).

[FIGURE 1 OMITTED]

During the 4-wk rearing period, in the starved animals the percentage of individuals with spent gonads increased, reaching about 50% after 2 wk, and as much as 90% after 4 wk. Relict gametes were often recovered, and ascini had thin walls with pale phagocytic meshwork. The percentage of individuals in which active gametogenesis continued (stages II-V) decreased, falling to about 5% after 4 wk. Specimens with gonads in recovery (stage I) also decreased to 5% after 4 wk of starving.

In contrast, in the fed animals, the percentage of individuals with active gametogenesis (stages II-V) remained constant (about 50%, increasing to 65% in sea urchins reared for 2 wk). Although there were no major spawning events, minor events were observed in the tanks in the second fortnight of the trial. As a consequence, the percentage of spent animals increased to 38 % during the 4 wk. Specimens with gonads in recovery always accounted for 10-12% of the total.

[FIGURE 2 OMITTED]

In sea urchins collected in June (corresponding to the end of the trial), various maturation stages were again observed. Compared with field t0 samples, however, a greater percentage of sea urchins with gonads in recovery (stage I) was recorded (30%), whereas active gametogenesis (stages II-V) was observed in about 20% of the sampled urchins.

Gamete Evaluation

Table 3 shows the number and relative percentages of sacrificed urchins in which visually assessed high gamete quality was observed, and the results of the fertilization tests (as percentages of normal developed plutei larvae obtained 72 h after fertilization). Viable gametes were mainly collected from individuals in the premature to spawning gonad stages (stages III-V), but spent gonads also frequently contained unspawned motile spermatozoa or morphologically intact eggs. In the field to collected sea urchins (May), 52.9% of male and 69.2% of female gonads were ripe, viable gametes. Four weeks later, these percentages were lower in field 4-wk samples (June), but were similar in fed animals. Viable gametes were collected in only 35% of starved animals. Sea urchin origin (rearing trials or natural conditions) significantly affected fertilization ability ([F.sub.2, 15] = 35.493, P < 0.001). Gametes from both field-collected and fed/ reared urchins yielded normally developed plutei larvae in about 80% of cases, whereas in the starved animals, the fertilization success was significantly lower (P < 0.001, Tukey's test).

[FIGURE 3 OMITTED]

The sperm motility parameters recorded by the SCA system in both wild-caught sea urchins and those reared for 4 wk are shown in Figures 4 and 5. The rapid sperm ranged around 80% and the total motile ones around 90%. As measured by 1-way ANOVA ([F.sub.2, 51] = 1.99, P = 0.147; [F.sub.2, 51] = 2.43, P = 0.098 respectively), neither the percentage of total motile spermatozoa nor the rapid spermatozoa showed any significant difference in relation to sea urchin origin (rearing trials or natural conditions). The recorded VCL ranged from 200-380 [micro]m/sec, the VAP ranged from 200-300 [micro]m/sec, and the VSL ranged from 100-280 [micro]m/sec. Again, the Kruskal-Wallis ANOVA test found no significant differences in the 3 groups of analyzed male urchins: [H.sub.2, 54] = 3.145, P = 0.207 for VCL; [H.sub.2, 54] = 3.744, P = 0.153 for VAP; [H.sub.2, 54] = 3.298; P = 0.192 for VSL.

DISCUSSION AND CONCLUSIONS

The annual reproductive cycle of P. lividus in the Mediterranean area is characterized by a long period in which the gonads have developing, mature, and spawning gametes (stages II-V according to Byrne (1990)). This lasts from September to May (Lozano et al. 1995), but can be longer in the case of particularly favorable environmental conditions, to the point that some individuals remain mature all year, with a brief resting period (stages VI-I) from July to September (Sanchez-Espana et al. 2004, Barbaglio et al. 2007). The specimens used in this experimental trial, taken from the mid to low Adriatic, exhibit a similar reproductive pattern, with about 60 % of specimens already in stages V-I in May (t0) and nearly 80% in June (field, 4 wk).

[FIGURE 4 OMITTED]

When reared in the absence of food, P. lividus specimens rapidly progressed in their reproductive cycle. After 4 wk, more than 90% of them were in the spent stage (stage VI), and although relict gametes were found in the gonads, no developing gametes were observed, as seen by Bishop and Watts (1994) in the case of the sea urchin L. variegatus. In addition, the GI of the starved animals halved, as seen in P. lividus and other species of sea urchins (Lares & Pomory 1998, Guillou et al. 2000, Arafa et al. 2006), which was the result of both the disappearance of gametes and the thinning of the phagocytic meshwork. Very little histological change was observed during the first 2 wk of starvation, with a more rapid change during the subsequent 2 wk. All animals may suffer periods of lack of food and may use different physiological strategies to cope with this (McCue 2010). Periods of more or less prolonged starvation are common events for sea urchins and, to a certain extent, they can model their biochemistry and physiology to remedy this (Lares & Pomory 1998, Russell 1998, Arafa et al. 2006).

In contrast, in the specimens fed for 4 wk, developing gametes were always present along the walls of the gonadal follicles, and new gametes were starting the maturation cycle while mature ones were spawning. At the same time, the GI greatly increased as a result of the dense network of phagocytes in specimens in stage II, and the presence of mature gametes in specimens in stages III and IV.

[FIGURE 5 OMITTED]

The capacity of P. liridus to produce gametes continuously, "skipping" the resting period, has been demonstrated in both natural populations living in environments characterized by high availability of food (Byrne 1990, Spirlet et al. 1998) and in closed-circuit cultivation conditions (Spirlet et al. 2000). It may thus be supposed that a similar effect occurred during our experimental conditions. Thus, unlike the starved specimens, the switch to rearing conditions that were more favorable than those of the natural environment produced in the fed sea urchins an immediate metabolic response, with the resumption of gametogenesis, as hypothesized for Strongylocentrotus droebachiensis by Russell (1998), in response to a sudden increase in the quality of food.

The amount, and the relative quality, of the gametes recovered in the reared urchins clearly depends on the gonad stage. However, it is worth noting that unspawned gametes were often also recovered from stage I (recovery) gonads. On the other hand, similar or longer starvation periods tested in L. variegatus (Bishop & Watts 1994) and S. granularis (Guillou et al. 2000) did not lead to total gonad regression. This is probably the result of the slowness and complexity of the gamete reabsorption process with respect to the shortness of the starvation period (Reunov et al. 2004).

Sea urchin sperm velocity parameters have been investigated by computer-assisted systems (Bracho et al. 1997, Au et al. 2001, Wood et al. 2007), and for the sea urchin Anthocidaris crassispina, VAP and VCL were found to be significantly correlated with fertilization rates. Indeed, VAP and VCL proved to be better predictors of fertilization than the percentage of motile spermatozoa (Au et al. 2002). The motion parameters of the P. lividus sperm recorded in this study by SCA showed levels comparable with those recorded by other computerized analysis systems in other sea urchin species (Bracho et al. 1997, Au et al. 2001, Wood et al. 2007). No differences were recorded in the sperm motility patterns of the sea urchins belonging to the 3 experimental groups, so we may suppose that the unspawned sperm cells found in recovery gonads are viable until they are phagocytosed (Reunov et al. 2004).

In contrast, the percentage of normal plutei larvae was significantly lower (P < 0.001) when gametes came from starved urchins. Because egg yolk granuli play a key role during larval development, serving not only as a source of nutrients (Unuma et al. 2009, Fujiwara et al. 2010), it may be supposed that even though the eggs of starved urchins seemed normally shaped in many cases, the processes of autolysis leading to gamete reabsorption (Byrne 1990) had already affected their biochemical composition.

Although temperature and photoperiod are important triggers of the gametogenic cycle in sea urchins (Walker & Lesser 1998, Pearce et al. 2004, Bruger et al. 2006), it has been extensively demonstrated that artificial diets can lead to a considerable increase in their gonad yield (Cook et al. 2000, Fernandez & Boudouresque 2000, Lawrence et al. 2003, McBride et al. 2004). Proteins are considered the main dietary component responsible for gonad production (Schlosser et al. 2005, Jacquin et al. 2006, Cook & Kelly 2007). The feed provided in this experiment had a significantly higher protein content than algae of the Ulva genus (about 25-27% according to Ortiz et al. (2006) and Valente et al. (2006)), which characterize the seabed where the sea urchins used in this trial were gathered.

Our results lead us to conclude that although the P. lividus specimens were gathered at the end of their reproductive season, when the process of recovery would be faster, the urchins reared under the experimented conditions immediately responded to the modified environmental conditions by reactivating gamete production, with no need to subject them to the stress of altering the photoperiod, and at the end of the 4-wk trial, viable gametes were recovered in almost 65% of the fed specimens. In addition, the closed rearing systems are laboratory scaled and easy to manage at some distance from the sea, and the formulated feed is not expensive, has no need of manipulation, and produces a negligible amount of waste. Thus, this can be considered an interesting short-term procedure for roe enhancement and, above all, for the continuous production of viable gametes, particularly for specimens from areas in which the environmental conditions allow for slow gametogenesis and, consequently, for a short reproductive season.

ACKNOWLEDGMENTS

This research was supported by MIUR-Italian Ministry for University and Research (MIUR Decr. 09.10.02; grant no. 143 to A. F.). Mr. George Metcalf revised the English text.

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ADELE FABBROCINI * AND RAFFAELE D'ADAMO

Consiglio Nazionale delle Ricerche-Istituto di Scienze Marine, UOS Lesina, via Pola 4, 71010 Lesina

(FG), Italy

* Corresponding author. E-mail: adele.fabbrocini@fg.ismar.cnr.it
TABLE 1.
Composition of the commercial formulated feed
(Classic K, hendrix, SpA).

                Dry Mass (%)

Crude protein       46.5
Crude fat           10.5
Crude fiber          2.4
Ashes                9.5

Proteins of animal origin account for less than 5%.

TABLE 2.
Gonad indices ([+ or -] SD) of reared urchins and of urchins
collected in the field on t0 and at the end of the rearing
trial (4 wk).

                      Males                Females

Field, t0       6.26 [+ or -] 3.10    6.96 [+ or -] 3.68
Field, 4 wk     4.88 [+ or -] 1.54    5.30 [+ or -] 2.56
Fed, 4 wk       9.74 [+ or -] 5.07   10.72 [+ or -] 3.71
Starved, 4 wk   3.66 [+ or -] 1.73    3.90 [+ or -] 1.56

                       Total

Field, t0        6.74 [+ or -] 3.75
Field, 4 wk         5 [+ or -] 1.98
Fed, 4 wk       10.23 [+ or -] 4.39
Starved, 4 wk    3.78 [+ or -] 1.62

TABLE 3.
Gamete quality evaluation of reared urchins and of urchins
collected in the field on t0 and at the end of the rearing
trial (4 wk).

                         Males

                Tot   HGQ   HGQ    Tot
                (n)   (n)   (%)    (n)

Field, t0       17     9    52.9   13
Field, 4 wk     19     9    47.4   11
Fed, 4 wk       14    10    71.4   16
Starved, 4 wk   14     5    35.7   16

                          Females

                HGQ   HGQ
                (n)   (%)          PL (%)

Field, t0        9    69.2   79.54 [+ or -] 3.16
Field, 4 wk      5    45.4   78.13 [+ or -] 4.24
Fed, 4 wk       10    62.5   79.86 [+ or -] 5.08
Starved, 4 wk    6    37.5   57.06 [+ or -] 7.01

HGQ, high gamete quality; specimens with a positive gamete visual
evaluation; PL, percent of normal developed plutei larvae (72 h
after fertilization); Tot, total analyzed specimens.
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