Formulated Diets Drive Gonadal Maturity but Reduce Larval Success in Paracentrotus lividus

Abstract

Over the past few decades, demand for sea urchin roe has risen, while wild sea urchin populations have declined. This trend has increased interest in aquaculture techniques and the development of formulated feeds to support ecological restoration and research. Here, we examined the effects of a high-protein formulated feed on gonad development in Paracentrotus lividus, compared to fresh feeds (maize and spinach), across three replicated tanks. We assessed gonad maturation, gamete viability, and larval development, and developed a new histopathological index applicable to both sexes. Formulated feed significantly enhanced gonad maturation, increased gamete production, and led to heavier gonads with higher gonadosomatic indices compared to fresh feeds, which were insufficient to promote maturation within four weeks. Notably, no histological alterations were observed in the gonads. Fertilization trials showed that embryos were produced, but none reached the pluteus stage, indicating decreased embryo viability. Although the formulated feed improved gonad development, it adversely affected water quality, increasing nutrient concentrations and lowering pH. Overall, these findings suggest that high-protein formulated feeds could potentially improve aquaculture production by enhancing gonad maturation and gamete output, but additional measures may be needed to support complete larval development.

1. Introduction

The reddish-orange gonads of the edible sea urchin Paracentrotus lividus, commonly called “roes”, are a prized delicacy along the Mediterranean coast, with consumption dating back to ancient Greece [1]. Since the early 1970s, global demand for sea urchin roe has increased, especially in Japan, which annually imports 20,000 tonnes, while its local production accounts for only 15,000 tonnes [2]. In Italy, Sardinia ranks among the regions with the highest consumption rates, with 30 million sea urchins (approximately 1800 tonnes) consumed annually, generating over 10 million euros of income [3]. This intense market demand has contributed to a significant decline in wild populations along the Mediterranean coast.

From an ecological perspective, P. lividus plays a significant role in structuring benthic communities, such as seagrass meadows and kelp beds, and in the recolonization of rocky substrates [4,5]. These ecological services underline the species’ economic value, which could be lost without adequate conservation measures [6]. Consequently, human activities affecting sea urchins and their habitats should be further regulated and combined with restoration initiatives. Sea urchin aquaculture provides a sustainable strategy to meet market demand while alleviating pressure on wild populations [7,8,9]. In the Mediterranean, two main approaches are used: land-based systems, including tanks and recirculating aquaculture systems (RAS), which allow precise control of temperature, diet, and stocking density, and sea-based grow-out structures, such as lantern nets and suspended cages, which have shown viable survival and gonad enhancement under operational conditions. These approaches demonstrate that P. lividus aquaculture is feasible and already practiced at various scales.

Optimizing diets is central to successful aquaculture because diet composition directly affects gonad maturation, gamete viability, and tissue quality [10,11].

P. lividus is mainly herbivorous but opportunistically consumes diverse foods in captivity, including algae, agar-based feeds, maize, spinach, and protein pellets [12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]. Protein-rich diets can improve gonad size and quality [24,28,29,30], yet excessive protein, especially of animal origin, can compromise gamete quality and reproductive success [28,29]. Maintaining both gonad growth and gamete viability is particularly critical for aquaculture aimed at scientific research or ecological restoration, where high-quality gametes are essential for fertilization and larval development [31,32].

Previous research indicates that pre-hydrated formulated feeds (e.g., Classic K^®; Hendrix pelletized compound feed) can increase gonad size over three months of feeding [24]. Despite this growth, gonads often fail to produce viable gametes, showing tissue hypertrophy and vacuolization, which leads to low fertilization rates and impaired embryonic development. These findings highlight the importance of assessing not only gonad size but also gamete viability when evaluating feeding strategies.

In this study, we investigated the effects of a high-protein formulated feed on the short-term gonad maturation and fertility of P. lividus, starting from spent-stage gonads. We compared compound feeds with traditional fresh feeds (maize and spinach) to evaluate their impact on gonad development, gamete viability, and potential applications in aquaculture and restoration programs.

2. Materials and Methods

2.1. Experimental Plan

A pool of 150 adult sea urchins was collected in the bay of Naples in February, by scuba divers of the Stazione Zoologica Anton Dohrn, when they were fully mature in their natural period of reproduction. The collection was set to ensure a sufficient number of males and females exhibiting a test diameter between 4 and 6 cm. Their sex was determined using a non-invasive technique based on the secondary sexual characteristics of genital papillae [33]. After selection of size classes and rearing of males and females in different 400 L tanks with recirculated seawater, sea urchins were starved 14 days prior to start the feeding experiment, to lead their gonads to a “spent” stage [34] evaluating the power of the formulated feed to promote an early gonad maturation, as compared to controls. The experiment took place in February 2022 in a temperature-controlled chamber set to 18°C ± 1 °C. Six independent closed circuit tanks, each with a capacity of 50 L, were used as microcosms (3 for the pellet treatment, 3 as controls). The tanks were filled with seawater pumped from the coast of Procida (Bay of Naples) and equipped with mechanical (2.5 Lt of synthetic sponge, corresponding to 5% of the tank volume) and biological (7.5 Lt of ceramic rings, corresponding to 15% of the tank volume) filters, as well as a water pump (600 L/h). An air stone connected to an air pump was also placed in each tank to ensure water circulation and maintain dissolved oxygen levels at saturation. A bacterial activator was added to each tank one week before the experiments and it promoted the maturation of the filter beds on ceramic rings. This one-week conditioning period facilitated biofilter maturation because we adopted Amtra Procult Filterstart (Amtra, Castronno, Italy) fast growing bacteria, according to the instructions of the manufacturer. After the initial bacterial colonization, nitrogen concentrations were kept constantly low also through the scheduled water changes regularly performed during the experiment. After a week, nine adult sea urchins (six females and three males with test size comprised between 4.5 and 6.3 cm) were weighed and placed in each tank. The number of tested females (6) in each tank was higher than the number of males (3) because at the moment of fertilization a prompt emission of males is normally observed while some females may not respond to the stimulation or produce insufficient amounts of eggs. At the start of the experiment the average weight of sea urchins placed in treatment tanks was 50.23 (±14.5) g, and 48.7 (±9.4) g for control sea urchins (

Table 1. (A) Composition and (B) nutritional profile of the protein pellet according to the manufacturer (Greenvet).

(A)
Flour proteins	11.00% (including 5% from 99% pure Spirulina)
Fishmeal	33.00%
Probiotics	10.00%
Insect flour from Tenebrio molitor	10.00%
Fish oil from salmon and cod	12.00%
Yeast	10.00%
Maize starch	14.00%
(B)
crude proteins	41.50%
crude lipids	30.00%
crude ash	8.20%
crude fiber	2.10%
starch	14.00%
chlorine	1.20%
calcium	1.10%
sodium	1.10%
phosphorus	0.80%

).

Individuals exceeding our experimental needs were returned to the sea. When the feeding experiment started, sea urchins in three control tanks were fed on a mixture of maize and spinach (1:1 by fresh weight), as typically used in laboratory tests. The natural diet of P. lividus consists primarily of macroalgae, although they also ingest animal epiphytes and are consequently considered omnivores. In controlled laboratory conditions, fresh macroalgae are not always available year-round, and their nutritional composition can vary substantially depending on season, origin, and storage conditions. For this reason, the use of maize and spinach as a fresh diet follows a long-standing and well-documented practice in sea urchin feeding trials [17,26]. Both spinach and maize are nutrient-rich, readily available, inexpensive, easy to standardize, and maintain stable quality during short-term experiments. In the other three (treatment) tanks, the sea urchins were given experimental pelletized feed. In summary, the experiment comprised 3 control tanks where sea urchins were fed on fresh meals and three treatment tanks where sea urchins were fed on experimental feed, and each tank consistently contained 9 individuals of comparable size. All six tanks received 10 g of feed every day, regardless of the treatment, because previous research [24] indicated that adult sea urchins in this size range consume ad libitum less than 1 g of food per day. Although pelleted and fresh feeds differ in density and digestibility, we provided the same dry-weight quantity of feed to all treatments to ensure comparability of nutrient input and to avoid confounding effects due to unequal feed availability. Feed remaining on the bottom was cleaned up every day prior to the next administration. The three experimental tanks were treated as true biological replicates, with individuals randomly distributed among tanks to minimize potential tank effects on the experimental outcomes. The protein pellets, developed by Greenvet (www.greenvet.com), were modified from an existing pellet available for commercial aquaculture of carps, and they had the composition and nutritional profile reported in Table 1.

This formulation, characterized by a high protein content and an odor that attracts sea urchins, could serve as a new food source for cultured sea urchins [29]. The pellets were shaped as small cylinders approximately 3 to 4 mm in length with a diameter of 3 mm. The mixture of canned maize and frozen spinach (which was thawed prior to use) was offered raw. The feeding experiments were conducted for only one month. This short duration was specifically chosen to detect the early effects of the different diets on gonad maturation, thereby minimizing the potential bias introduced by long-term physiological adaptations [35] that might otherwise influence the results. In fact, longer tests (e.g., 3–4 months) may easily lead to gonad maturation with various feeds, including natural items [36]. Here, we aimed at establishing the actual efficacy of the feeds to promote an early development of gonads, specifically starting from a spent stage. The position of the treatment tanks in the thermostatic chamber was alternated with the control tanks, to minimize any spatial effect of temperature (18 °C), light (about 80 µE with a 12/12 light/dark photoperiod), or other unknown influencing factors [37]. Every other day, the water in each tank was analyzed and either partially or completely changed, depending on the results of the water analyses. The 30-day feeding period was selected to assess short-term gonad maturation starting from spent-stage gonads. This duration allowed us to observe the initial gonad responses while minimizing prolonged exposure that could introduce confounding physiological factors.

2.2. Fertilization

After four weeks of feeding, all nine sea urchins present in each tank were individually weighed, and their gametes were collected for in vitro fertilization. To avoid the influence of gut contents on the body weight, sea urchins were not fed for 24 h prior to weighing. To obtain the gametes, male and female sea urchins were initially subjected to vigorous shaking to test the effect of physical stress. If gametes were not released by shaking alone, 1 mL of 0.5 M KCl (standard concentration commonly applied for sea urchin gamete release [24]) was injected into the coelom through the soft tissue around the mouthparts to induce gonadal contraction. The sea urchins were then shaken vigorously again. Females were placed mouth-side up over 50 mL beakers until they released their gametes into filtered seawater (0.22 μm Millipore), facilitating the collection of oocytes. The oocytes were rinsed three times with clean seawater to remove any organic residues.

Sperms from males were collected “dry” using a Pasteur pipette, extracting them directly from the surface of the gonophores to prevent premature activation that could occur upon contact with seawater. For the fertilization, a drop of sperm was added to Petri dishes containing the eggs, and a sample was observed under an optical microscope to detect the formation of the fertilization membrane, which typically appears as a clear ring visible at low magnification after 40–80 s.

Approximately one-hour post-fertilization (hpf), the first cell division occurred. Fertilization success was assessed by counting fertilized embryos and calculating the fertilization rate from five counts per sample. The embryos were then incubated in a thermostatic chamber at 18 °C for 48 h until they reached the pluteus stage. Morphological malformations were assessed 48 hpf by examining at least 100 fixed sea urchin plutei (fixed with 0.5% glutaraldehyde) from each female under a light microscope (Zeiss Axiovert 135TV, Carl Zeiss, Jena, Germany). The examination focused on arm shape, body symmetry, swimming movements, and the presence of morphological abnormalities [38,39].

2.3. Water and Biotic Parameters

Water parameters in the experimental (pellet-fed) and control (spinach and maize-fed) tanks were monitored every three days for four weeks. Nutrient concentrations (NO₂, NO₃, NH₄, and PO₄) were measured using standard kits for an “AL450” photometer (Aqualytic, Mombasa Road, Kenya). Physical parameters such as temperature, salinity, pH, and dissolved oxygen (O₂) concentration were continuously measured using specific probes and adjusted as needed to meet the requirements of P. lividus [40,41,42]. The behavior, spawning, mortality, and health of the sea urchins were assessed every two days, as environmental factors can influence somatic growth and gonadal health [43]. Mortality (dead individuals were immediately removed), spine loss (indicative of disease), and abnormal behavior were visually checked in animals fed both on the control diet and the protein pellet. Throughout the experiment, survival, spine integrity, locomotion, and general responsiveness were monitored, and no signs of disease or stress attributable to the formulated feed were observed in any treatment.

2.4. Histological Analysis of Gonads

After four weeks of feeding treatments, gonads from fifteen sea urchins per treatment group were used for histological analysis. The peristomal membrane was incised to access the gonads, which were then blotted dry, fixed in modified Carnoy’s solution, and processed for histological examination using haematoxylin staining. The histopathological index, adapted from Costa et al. (2013) [44], was calculated using the following formula:

$${\mathrm{I}}_h ={\displaystyle \frac{\sum wa}{\sum M}}$$

where w is the weight of the selected histopathological alteration, a is the score attributed to the alteration based on its diffusion grade (a value from 0 to 6), and M is the maximum attributable value for the alteration (in the case that all alterations are present at the maximum diffusion grade). Six random images were taken of each gonad using a Canon PowerShot S50 camera connected to a Leica DM RB optical microscope via the RemoteCapture acquisition tool. These images were blindly examined to determine the diffusion score of each analyzed alteration [45]. The analyzed alterations included lipofuscin accumulation, detachment of acinar borders, and enlargement of interstitial spaces among gametes (Figure 1), with corresponding weights presented in

Table 2. Gonadic alterations analyzed for the development of a histopathological index in P. lividus.

Tissue	Histopathological Alteration	Weight (w)
Gonads	Lipofuscin aggregates	1
	Detachment of acinal borders	2
	Enlargement of interstitial spaces among gametes	1

, which were established according to their biological relevance. Lipofuscin accumulation, which was assessed as an index, represents a general marker of oxidative stress and cellular aging. Detachment of acinar borders was noted as a more severe lesion, reflecting the disruption of acinar structural integrity. Finally, enlargement of interstitial spaces was quantified as an index indicative of early tissue disorganization [44,45,46,47,48].

2.5. Statistical Analysis of Data

The gonadosomatic index (G.I.) was calculated as the ratio of fresh weight of gonads to the fresh weight of sea urchins. A paired t-test was used to determine the significance of differences in both biotic and abiotic parameters between experimental and control tanks. This technique was adopted because the experimental design involved strict pairing and co-location of control and treatment tanks, allowing the paired t-test to effectively remove the variability inherent to the tank-to-tank differences (noise) and maximize the statistical power to detect the true effect of the treatment. Homoscedasticity was checked by performing a Bartlett test. Additionally, a similarity matrix was produced to evaluate correlations among seawater parameters, considering Pearson’s correlation coefficients from ±0.4 to ±0.6 as moderate and from ±0.7 onwards as strong correlations. Histopathological indices were compared between pellet and control groups using the Mann–Whitney U test. Data analysis and graphing were performed using GraphPad Prism ver. 9.0.

3. Results

3.1. Gonadosomatic Indices

The average weight of sea urchins at the end of the experiment was 48.9 (±9.4) g for control individuals and 45.8 (±12.01) g for treatment ones, showing that the average weight was not significantly changed after 1 month of rearing (

Table 3. Sea urchin weights (in grams = g) at the beginning and at the end of the experiment. See the average values and standard deviations in the lower part of the tables.

Control sea Urchin Weights (g) (Maize and Spinaches)			Treatment Sea Urchin Weights (g) (Protein Pellet)
Beginning of the experiment
Tank V1	Tank V3	Tank V5	Tank V2	Tank V4	Tank V6
55.3	48.38	59.25	70.65	45.12	82.25
48.37	57.27	66.12	58.23	48.83	47.3
49.95	45.43	67.89	75.9	34.91	46.34
57.21	59.9	38.48	63.57	39.03	37.63
47.06	47.1	48.77	55.89	54.05	72.11
47.54	33.31	41.45	52.67	34.7	54.29
43.74	29.9	46.67	48.18	25.57	60.99
56.09	32.93	50.83	27.56	30.46	52.98
50.15	44.76	40.1	49.35	40.32	47.46
Average: 50.23 (±14.48) g			Average: 48.7 (±9.4) g
End of the experiment
50.1	35.2	42.2	71.2	53.9	44.7
54.5	49.6	54.2	50	33.4	62.5
47.3	48.7	50.4	55	436	54.7
51.7	53.7	65.3	48.2	31.9	44
59	59.1	68.5	28.6	43.9	49
58	46	58.8	54.7	39.2	57.2
46.6	35.3	41.5	52.2	25.1	40.1
46.1	31.7	48	70.9	38.6	72
47.8	31	41.8	64.5	34	80.5
Average: 48.9 (±9.4) g			Average: 45.8 (±12.01) g

).

Similarly, differences in test size between control and treated sea urchins, both at the beginning and the end of the experiment, were not significant. However, gonads extracted from sea urchins fed on protein pellets had an average weight significantly higher than those from sea urchins fed on fresh items (

Table 4. Gonad weights (in grams. g) for sea urchins in control tanks (V1, V3 and V5) and treatment tanks (2, 4 and 6). See the average values and standard deviations in the lower part of the table. N.d. = not determined.

Gonad Weight (g) Control			Gonad Weight (g) Treatment
(Maize-Spinaches)			(Protein Pellet)
Tank V1	Tank V3	Tank V5	Tank V2	Tank V4	Tank V6
2.49	2.47	2.5	3.86	0.56	1.33
2.5	2.47	2.5	1.02	1.65	3.23
2.47	2.48	2.51	1.25	2.69	1.61
2.5	2.49	2.51	1.3	1.08	2.22
2.49	2.5	2.5	1.31	1.20	1.24
2.51	2.47	2.46	0.93	2.49	1.86
2.5	2.48	2.49	2.35	1.86	1.86
2.55	2.55	2.52	1.41	0.6	1.7
2.51	2.55	2.5	1.70	n.d.	n.d.
Average: 1.69 (±0.78) g			Average: 2.50 (±0.02) g

).

Differences between the two groups were significant (t-test; p < 0.001; Table S1).

G.I.s for sea urchins fed on protein pellets were significantly higher than those fed on fresh items (Figure 2,

Table 5. Gonadosomatic indices (G.I.s) for sea urchins in control tanks (V1, V3 and V5) and in treatment tanks (V2, V4 and V6). See the average values and standard deviations in the lower part of the table. N.d. = not determined.

G.I. Control			G.I. Treatment
(Maize-Spinaches)			(Protein Pellet)
Tank V1	Tank V3	Tank V5	Tank V2	Tank V4	Tank V6
3.50	4.58	5.59	7.71	1.61	3.16
5.00	7.40	4.00	1.87	3.33	5.97
4.49	5.69	4.59	2.66	5.53	3.2
5.19	7.81	5.70	2.51	2.02	3.41
8.71	5.69	5.10	2.22	2.03	1.81
4.59	6.30	4.30	1.61	5.42	3.16
4.79	9.88	6.21	5.05	5.27	4.48
3.60	6.61	3.50	3.08	1.9	3.54
3.89	7.50	3.11	3.56	n.d.	n.d.
Average: 3.44 (±1.60)			Average: 5.46 (±1.68)

). Gonadosomatic indices (G.I.) were compared among treatments and vs. the control tanks and they exhibited statistically significant differences (p < 0.01; Table S2).

3.2. Water Quality

Water parameters for three experimental tanks (

Table 6. Water parameters for the three experimental tanks (sea urchins were fed with pellet) measured every three days for four weeks.

	NH4	NO2	NO3	PO4	O2	Salinity	T	pH
0	1.53	0.00	10.03	0.24	5.31	40.00	17.73	7.70
1	1.73	0.00	9.17	0.24	5.70	41.33	17.33	7.74
3	1.73	0.00	11.27	0.24	5.90	41.00	16.73	7.88
7	2.27	0.00	6.73	0.19	5.97	40.33	16.40	7.94
9	2.90	0.00	6.73	0.19	5.78	39.83	17.03	7.84
11	2.90	0.00	6.73	0.16	5.21	40.33	17.65	7.63
16	2.77	0.03	6.73	0.31	3.83	41.00	17.40	7.84
18	2.63	0.04	6.73	0.31	5.52	40.67	17.53	7.89
21	3.07	0.03	6.73	0.39	5.49	40.67	17.63	7.77
23	3.07	0.03	6.73	0.39	5.07	40.67	17.67	7.88
25	3.37	0.05	6.73	0.48	5.46	4067	17.60	7.91
28	2.95	0.05	3.90	0.34	5.32	40.00	17.85	7.90

, sea urchins fed on pellet) and control tanks (

Table 7. Water parameters for the three control tanks (sea urchins were fed with spinach and maize) measured every three days for four weeks.

	NH4	NO2	NO3	PO4	O2	Salinity	T	pH
0	0.74	0.00	9.27	0.09	5.08	40.00	17.53	7.70
1	0.84	0.00	9.33	0.09	5.56	40.33	17.13	7.80
3	1.97	0.00	11.93	0.09	5.90	40.00	16.63	7.95
7	0.77	0.00	11.10	0.09	6.02	40.00	16.53	8.00
9	0.77	0.00	11.10	0.09	5.11	39.50	17.43	7.75
11	2.44	0.01	11.10	0.15	5.21	40.67	17.50	7.72
16	3.07	0.03	11.03	0.15	5.55	41.00	17.73	8.03
18	3.10	0.03	11.03	0.15	5.53	41.33	17.67	7.89
21	2.33	0.01	11.10	0.17	5.18	41.33	17.63	7.85
23	2.33	0.01	11.10	0.17	5.39	40.67	17.53	7.93
25	2.57	0.01	11.10	0.21	5.28	40.33	17.53	7.88
28	2.90	0.01	7.20	0.25	5.05	40.00	17.80	7.70

, sea urchins fed on fresh items) were measured and the significance of differences were validated by paired t-test between the measures in the two conditions are reported in Table S3.

Significant differences were detected in NH₄ concentration among the replicate tanks containing sea urchins fed on pellets, ranging from 1.53 to 3.37 ppm (2.57 ± 0.6). In control tanks (sea urchins fed on fresh items) NH₄ ranged between 0.74 and 3.10 ppm (1.98 ± 0.94) and the differences with treated tanks were statistically significant (p < 0.05, Table S3). A gradual increase in NO₂ was detected in the treatment tanks, reaching 0.05 ppm at the end of the experiment (0.019 ± 0.02), while it slightly increased from the 11th to the 18th day in control tanks, and then progressively decreased until the end of the experiment (0.001 ± 0.001). The differences between control and treatment tanks were not significant (p > 0.05). In the treatment tanks the concentration of NO₃ ranged between 3.90 and 11.27 ppm (7.3 ± 1.9), while in the control tanks it ranged from 7.20 to 11.93 ppm (10.5 ± 1.3) (p < 0.001). The concentration of PO₄ in the treatment tanks ranged between 0.16 and 0.48 ppm (0.29 ± 0.02), while in the control tanks the values progressively increased from 0.09 to 0.25 (0.14 ± 0.05) (p < 0.0001). The concentration of dissolved oxygen in the treated tanks ranged between 3.83 and 5.97 ppm (5.38 ± 0.56), while in the control tanks it ranged from 5.05 to 6.02 (5.4 ± 0.31). No significant differences were found between the two experimental conditions (p > 0.5). Salinity showed a similar trend in the experimental tanks (from 39.83 to 41.33 PSU; 40.5 ± 0.45) and in control tanks (from 39.5 to 41.33 PSU; 40.43 ± 0.58). Consequently, the differences between control and treatment tanks were not significant (p > 0.05). Temperature was approximately the same in both treated and control tanks, ranging from 16.40 to 17.85 °C in the treated tanks (17.37 ± 0.44 °C) and from 16.50 to 17.80 °C in the control tanks (17.38 ± 0.41 °C), with no significant differences found (p > 0.5). Similarly, pH ranged from 7.63 to 7.94 in the treatment tanks (7.82 ± 0.09) and from 7.70 to 8.03 in the control tanks (7.85 ± 0.11). The differences in pH between control and treatment tanks were also not significant (p > 0.05). In the treatment tanks, a positive correlation was detected between NH₄ and NO₂ (0.667), and NH₄ and PO₄ (0.621), as well as a negative strong correlation between NH₄ NO₃ (−0.715). In contrast, a positive correlation was found between i. NO₂ both with pH (0.522) and temperature (0.456); ii. NO₂ with PO₄ (0.847); iii. PO₄ with temperature (0.428); iv. NH₄ with NO₂ (0.933) and temperature (0.715), as well as with PO₄ (0.655) and salinity (0.663); v. NO₂ with salinity (0.832), temperature (0.846) and PO₄ (0.641); vi. NO₃ with temperature (−0.602); vii. PO₄ with salinity (0.461) and temperature (0.557); vii. dissolved O₂ concentration with pH (0.936); viii. salinity with temperature (0.717). A negative correlation was also detected between NO₂ with NO₃ (−0.646), which in turn was negatively correlated with the pH (−0.415); while dissolved O₂ showed a strong negative correlation with temperature (−0.734).

3.3. Histopathological Analyses

The histological analysis revealed that treated sea urchins displayed various stages of gonadal development. Some pellet-treated females exhibited previtellogenic oocytes in development (Figure 3A,B), while others showed mature oocytes in the gonoducts or residual oocytes in the center of the acinus (Figure 3C,D). Treated males were generally observed in either a post-spawning or developmental stage, with spermatocytes located along the cortex of the acinus (Figure 3E,F).

In comparison, control females also displayed a predominantly refractory phase, with some previtellogenic oocytes positioned along the cortex (Figure 4A,B) and a few atretic previtellogenic oocytes (Figure 4D). Only one control female showed a few mature oocytes (Figure 4E). Similarly, control males were mainly in a post-spawning stage, with residual spermatozoa or spermatocytes along the border of the acinus (Figure 4C,F).

Overall, the histopathological index indicated no significant differences between the two treatments (Figure 5).

3.4. In Vitro Fertilization

Sea urchins fed on the formulated feed in the treatment tanks yielded two females and two males emitting mature gametes upon injection of 1 mL of 0.5 M KCl. In contrast, in the control tanks there were no females emitting gametes upon KCl injection, and only two males emitted spermatozoans after stimulation. No developmental data could be obtained for the control group, as none of the females fed on fresh items released mature oocytes upon KCl stimulation, preventing fertilization assays and subsequent embryonic assessment.

Mitotic divisions occurred in several embryos produced by sea urchins fed on pellets (treatment), but their phenotypes were irregular (Figure 6 and Figure S1), and several embryos exhibited a darker content. As regards the remaining embryos deriving from the females fed on pellets, no embryos reached the pluteus stage 48 hpf. All embryos exhibited delayed development and were malformed, remaining at the blastula, gastrula, prism, or early pluteus stages. Fertilization outcomes are presented exclusively for the treated individuals, since the control groups lacked mature oocytes, thereby preventing the assessment of embryo development.

4. Discussion

The depletion of wild stocks in the Mediterranean increased the need for sustainable aquaculture to supply high-quality sea urchin roe. P. lividus, often classified as an opportunistic omnivore, shows clear feeding preferences influenced by the chemical and physical properties of food [49,50,51,52]. Despite being primarily herbivorous, its feeding behavior changes based on food availability, and it also shows higher consumption rates after starvation periods [53]. Given the slow growth rates of this species [54] and the significant relationship between growth and food quality and quantity [55], this research focused on the impact of a protein-based diet on gonadal production and gamete maturation rather than on size increments. The study of the effects of experimental feeds on its physiology and the reproductive success of sea urchins facilitate understanding of their ecological success and the development of new feeds for echinoculture practices [56]. The experimental diet aimed at enhancing gonadal production and gamete maturation, critical for the success of echinoculture practices [34].

Our experimental setup aimed at comparing sea urchins fed on protein pellets to those fed on fresh items (spinach and maize). The latter were chosen as controls due to their nutrient-rich composition and extensive testing in previous studies [17,57]. To standardize their physiological state, all individuals were starved before the experiment, because two weeks of starvation are known to induce a spent gonadal stage both in males and females of P. lividus [34]. Thus, any production of mature gametes at the end of the experiment should be considered to be due to recovery processes realized during the 30 days of forced feeding. This research also aimed at evaluating if a formulated diet could provide a fast recovery of gonads, up to maturation of gametes, after one only month of “ad libitum” feeding. A protein pellet, initially developed for carp aquaculture, was evaluated for its potential role in formulating a new feed for cultured sea urchins. This evaluation was based on its key attributes: high plant protein content, attractive odor, cost-effectiveness, and durability in seawater. We found that sea urchins fed on protein pellets had significantly heavier gonads and higher gonadosomatic indices (G.I.) compared to the control group (spinach and maize-fed).

Furthermore, istological analyses suggested that protein pellets may enhance gonadal maturation and gamete production. Females fed on pellets showed mature oocytes, and males produced mature spermatozoa, indicating potential gamete maturation. In contrast, control females remained in a refractory stage, with immature oocytes. Notably, only pellet-fed females emitted eggs, suggesting a nutritional advantage of the pellets over the fresh items. Additionally, the histopathological index, commonly applied to invertebrates to quantify tissue damage [45,58] was evaluated in both sexes. Since no morphological alterations of gonads were found in sea urchins treated with pellets, as compared to controls, the safety of this experimental feed is supported. Although pellet-fed sea urchins showed enhanced gonadal development and successfully produced mature gametes, the embryos derived from these gametes did not progress normally [59]. This result indicates that improved gonadal maturation did not translate into successful embryogenesis, highlighting a critical disparity in reproductive output. In this context, our research specifically investigated the effects of formulated feeds on gonadal maturation (through histological analyses) and fertility (through bioassays) under controlled conditions. The high protein content of the pellets likely contributed to nutrient leaching and consequent changes in water chemistry, which may in turn have negatively affected gamete viability and early development [60]. Thus, the observed reproductive outcomes likely result from an interaction between feed composition and water quality deterioration, rather than from diet composition alone. Consistent with previous work [37,43], we found that high-protein diets significantly increased the gonadal index and accelerated maturation. Unlike studies using long-term feeding (3–4 months), our results demonstrate that meaningful gonadal recovery can occur within as little as 30 days following starvation.

Research has already highlighted the importance of developing sustainable farming practices and larval production for restocking purposes in species like P. lividus. For instance, Gago et al. [60] observed that the amino acid composition of P. lividus eggs and larvae is largely unaffected by broodstock diet manipulations. More recently, Ciriminna et al. [61] explored the impact of sustainable diets on adult P. lividus females. These studies, along with the present research, underscore the critical importance of broodstock diet in influencing key reproductive parameters and gamete quality. Our results agree with studies reporting that protein-rich diets enhance gonadal size but do not necessarily produce viable gametes [29]. The present work strengthens this evidence by showing that even when histology indicates mature gametes, embryonic development may still be compromised. Future research will focus on in-depth biochemical analyses of eggs, including the presence and abundance of various types of vitellogenin (Vtg), to further enhance our understanding of these results and optimize breeding strategies for restocking programs.

Despite the positive effects on gonadal maturation, embryos from pellet-fed females showed delayed development and did not reach the pluteus stage. They exhibited irregular phenotypes, likely due to apoptosis, aligning with previous studies [24] that indicated how high-protein diets from plant sources increased gonadal indices but resulted in non-viable gametes. Our results also showed that embryos from females fed on pellets were delayed in their development. No embryos reached the pluteus stage; they were at the blastula, gastrula, prism, or early pluteus stages. Several embryos displayed irregular phenotypes and darker internal content, potentially due to advanced apoptosis processes. Since apoptosis was not directly measured in this study, we now refer to it only as a possible explanation rather than a confirmed mechanism for the observed embryo failure. While the darker appearance and irregular morphology of the embryos suggest compromised development, further cytological or molecular analyses would be required to definitively verify apoptotic activity. These findings align with previous studies [24], which indicated that a high-protein commercial feed led to increased gonadal indices but resulted in non-viable gametes.

Our results also indicated that protein pellets negatively influence water quality. Treatment tanks exhibited higher concentrations of NH₃, NO₂, and PO₄, and lower pH levels compared to control tanks. The high protein content of pellets caused nutrient leaching when not immediately consumed by sea urchins. These issues in water quality are consistent with conditions known to impair gamete quality, fertilization success, and embryo stability in echinoderms. This consistency provides a plausible environmental pathway contributing to the reduced embryo viability observed in the pellet-fed treatment. Correlation matrices showed that in control tanks, salinity and nutrient concentrations (NO₃, NH₃, NO₂, and PO₄) were mainly correlated with temperature, while these correlations were weaker in treatment tanks. At higher temperatures, control tanks experienced stress, whereas organic pollution was consistent in treatment tanks throughout the experiment. Importantly, even though changes in water chemistry were observed in the treatment tanks, no mortality or behavioral abnormalities occurred. Furthermore, histopathological analyses confirmed the absence of tissue damage, indicating that the formulated feed did not impair the general health status of the sea urchins during the 30-day trial.

Taking into account the negative impacts on water quality, the positive effects of pellets on the reproductive physiology of sea urchins are significant, especially given the short experimental period. The protein pellet is cheaper than fresh items, easy to store, and safer for the functionality of filters in a recirculating aquaculture system (RAS). It is also easier and quicker to administer, without causing water to darken. In contrast, spinach and maize require refrigeration, defrosting before administration, they rot quickly, and often cause mechanical issues in recirculating systems, such as clogging pumps and filters, and releasing pigments into the water.

While this 30-day study successfully assessed short-term gonadal recovery, further research is required to evaluate the long-term dietary effects on the full reproductive cycle and sustained gamete viability in this species. The formulated feed, originally developed for carp, may not have fully met the nutritional requirements of P. lividus, possibly contributing to the observed embryonic abnormalities. Moreover, nutrient leaching from the pellets affected water quality, introducing potential confounding factors. As the experiment was conducted under controlled laboratory conditions, the findings may not fully reflect responses under commercial or natural aquaculture environments. Future research should include longer trials using species-specific feed formulations and detailed biochemical analyses to better understand the mechanisms influencing gamete quality and embryo development.

In conclusion, formulated high-protein pellets successfully enhanced gonadal growth and maturation in P. lividus over a short feeding period. However, embryos derived from pellet-fed females showed delayed development and failed to reach the pluteus stage, indicating limited fertility. These results suggest that, while pellets may promote gonadal recovery, their effects on gamete viability are insufficient for ensuring successful reproduction. Therefore, further optimization of feed composition is needed before recommending widespread use in aquaculture or restorative programs.