AbstractMaintaining the reproductive performance of Litopenaeus vannamei broodstock toward the end of the breeding season remains a major challenge for commercial hatcheries, primarily due to physiological exhaustion. This study evaluated whether diversified dietary formulations could extend the breeding period and enhance larval quality. Broodstock (males: 50 ± 2 g; females: 69 ± 5 g) were allocated to four dietary treatments comprising worms (Nereis spp., ragworm), squid (Loligo forbesii), and mussels (Mytilus galloprovincialis). Group 1: 80% worms and 20% squid; Group 2: 80% worms and 20% mussels; Group 3: 50% worms, 25% mussels, and 25% squid; and Group 4: equal proportions (25% each) of worms, mussels, squid, and chicken liver. Over a 30-day trial, reproductive indices (HSI, GSI, OMI), hemolymph biochemistry, hepatopancreatic antioxidant status, and the expression of key maturation-related genes (vih, vtg, iag, igfbp1) were assessed, alongside histological examination of the hepatopancreas and gonads. Group 3 exhibited the highest reproductive efficiency, producing significantly greater egg output and larval yield compared with the worm-dominated control (Group 1). Molecular analyses showed a pronounced downregulation of vitellogenesis-inhibiting hormone (vih) in the eyestalks, accompanied by upregulation of vitellogenin (vtg) in the hepatopancreas of Groups 3 and 4. In males, expression of the insulin-like androgenic gland hormone (iag) peaked in Group 4, corresponding with enhanced histological structure of the vas deferens. Group 4 also exhibited enhanced hepatopancreatic antioxidant status, characterized by increased superoxide dismutase activity and reduced lipid peroxidation. Histological observations showed that diversified diets promoted healthy hepatopancreatic structure and sustained vitellogenic progression in the ovaries. Overall, reducing reliance on polychaetes and incorporating a balanced mix of mussels, squid, and chicken liver provides an effective nutritional strategy to rejuvenate aging broodstock. These findings offer a practical nutritional approach for hatcheries to enhance seed production and potentially prolong broodstock productivity, with the observed reproductive responses associated with changes in neuroendocrine-related marker gene expression.
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IntroductionOver the past several years, the global aquaculture industry has experienced significant progress, with whiteleg shrimp farming now accounting for more than 80% of total global penaeid shrimp production1,2. This growth has been driven largely by the combined influence of several key factors, most notably the adoption of improved strains of Pacific white shrimp (Litopenaeus vannamei). These advancements have resulted in higher survival rates, faster growth, reduced production costs, and greater resistance to disease3,4.As a consequence, demand for shrimp has risen substantially across both domestic and international markets, strengthening the industry’s role as a highly profitable and strategically important economic sector5,6. Over the past two decades, the cultivation of Pacific white shrimp has expanded rapidly, with the species being introduced to an increasing number of countries and its global production footprint continuing to grow2. This widespread adoption is largely due to its strong adaptability to a range of aquaculture environments, along with its relatively high tolerance to diseases and environmental stressors compared with other shrimp species7,8,9.This sustained growth has, in turn, intensified interest among researchers and investors in improving broodstock quality to maximize production. Commercial hatcheries rely on consistent broodstock performance to supply high-quality postlarvae for grow-out operations. However, maintaining reproductive performance over extended breeding seasons remains a major challenge10. As broodstock age and undergo repeated spawning cycles, physiological exhaustion, depleted energy reserves, oxidative stress, and endocrine dysregulation can reduce fecundity, egg quality, and larval viability—ultimately lowering seed output and increasing production costs11,12.Nutrition plays a central role in reproductive success in crustaceans. It provides the substrates required for gametogenesis, supports the synthesis of lipoproteins and yolk precursors, modulates antioxidant defenses, and interacts with neuroendocrine pathways that regulate maturation13,14. In penaeid shrimp, the hepatopancreas serves as the primary metabolic organ, storing and converting dietary lipids and proteins into vitellogenin and other compounds essential for oocyte development15. Consequently, diet strongly influences key reproductive indices—hepatosomatic index (HSI), gonadosomatic index (GSI), and ovary maturation index (OMI)—as well as molecular regulators such as vitellogenin (vtg), insulin-like androgenic gland hormone (iag), and neuropeptides including vitellogenesis-inhibiting hormone (vih), produced in the eyestalk X-organ–sinus gland complex16,17,18.Although formulated broodstock diets have been developed and are increasingly incorporated into maturation protocols, commercial shrimp hatcheries continue to rely heavily on fresh or frozen marine ingredients19,20. Traditionally, polychaete worms have been widely used as a live or fresh feed for broodstock due to their high protein and essential fatty acid content, as well as their ability to stimulate spawning and improve larval quality21,22. However, prolonged reliance on a single feed source can be limiting. Monodiets often fail to provide a balanced supply of long-chain polyunsaturated fatty acids (LC-PUFAs), sterols, phospholipids, micronutrients, and other conditionally essential compounds required for sustained vitellogenesis and antioxidant defense. This imbalance can lead to physiological depletion and reduced reproductive output, necessitating dietary supplementation23,24.Among the fresh feed ingredients available for broodstock nutrition, mussels, squid, and chicken liver are of particular interest because their distinct nutrient profiles may address different physiological requirements associated with gonadal maturation and repeated spawning. Mussels are rich in phospholipids and micronutrients that may support hepatopancreatic function and vitellogenesis, whereas squid provides highly digestible protein and docosahexaenoic acid (DHA), both of which contribute to egg membrane integrity and larval neural development25,26,27. Chicken liver is a concentrated source of cholesterol, retinol, and B vitamins, which play important roles in steroidogenesis and energy metabolism during successive spawning cycles28,29. Chicken liver and enzymatically processed chicken liver powder have also been evaluated as alternatives to fishmeal in diets for juveniles Pacific white shrimp, with studies reporting improvements in growth and immune-related parameters30,31. Compared with diets based on a single fresh feed ingredient, combining these nutritionally distinct sources may provide a more balanced supply of essential proteins, lipids, vitamins, and micronutrients. This complementary nutritional profile provided the rationale for evaluating mussels, squid, and chicken liver as dietary components for sustaining broodstock reproductive performance throughout an extended breeding period.Beyond macronutrient supply, diet also influences oxidative status and the ability of broodstock to withstand oxidative stress associated with intense metabolic activity during gametogenesis and spawning32. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) play a critical role in neutralizing reactive oxygen species (ROS)33,34. These ROS may be generated during yolk formation and embryogenesis32,35. Insufficient antioxidant protection can lead to lipid peroxidation in oocyte membranes and reduced larval viability32,35. Diets enriched with antioxidant vitamins (A and E), selenium, and related cofactors therefore have strong potential to preserve gamete integrity and extend the reproductive lifespan of broodstock36. Consistent evidence in shrimp and other crustaceans links enhanced dietary antioxidant capacity with improved egg quality and larval survival36,37.Reproductive regulation in crustaceans is governed by a tightly integrated endocrine axis that links environmental and nutritional cues to the molecular processes controlling maturation. The eyestalk-derived neuropeptide vih acts as a key inhibitor of vitellogenesis; reduced vih activity permits increased synthesis of vitellogenin, the primary yolk precursor17. In contrast, the androgenic gland hormone (iag) drives male sexual differentiation and supports reproductive function, including sperm maturation and mating success38,39. Nutritional inputs can influence the expression of these regulatory pathways, modulating hormones such as estradiol, vitellogenin, luteinizing hormone, follicle-stimulating hormone, and testosterone40.Consequently, optimizing fresh-feed combinations remains highly relevant to commercial broodstock management. The present study evaluated whether reducing reliance on polychaetes and introducing diversified diets containing different proportions of worms, mussels, squid, and chicken liver could extend the effective breeding period of L. vannamei broodstock toward the end of the reproductive season.Chicken liver was included as an exploratory, nutrient-dense animal ingredient to increase dietary diversity, given that terrestrial animal tissues have previously been used in shrimp maturation diets as supplementary sources of cholesterol, phospholipids, and micronutrients41. The 80:20 formulations maintained a consistent worm-based reference while allowing the effects of individual supplementary ingredients to be compared. In contrast, the 50:25:25 and equal 25% formulations represented intermediate and extensive levels of worm replacement, respectively. These treatments followed a stepwise ingredient-substitution approach commonly used in broodstock nutrition trials. Thus, the selected proportions established a controlled gradient of dietary diversification and ingredient replacement rather than representing previously established nutritional optima41.We hypothesized that mixed diets providing complementary sources of protein, long-chain polyunsaturated fatty acids, sterols, phospholipids, and antioxidants would: (1) sustain or improve the hepatosomatic index, gonadosomatic index, and overall reproductive performance; (2) maintain or improve hemolymph biochemical parameters related to lipid metabolism, energy status, and reproductive physiology; (3) enhance antioxidant enzyme activity while reducing lipid peroxidation; (4) shift neuroendocrine gene expression toward reproductive activation, as indicated by the downregulation of vih, upregulation of vtg, and modulation of iag and igfbp1 in males; and (5) support hepatopancreatic recovery and continued vitellogenic progression, as evidenced by ovarian histology, relative to a worm-dominated control diet. Demonstrating these effects would provide hatcheries with practical dietary strategies for sustaining the reproductive capacity of aging broodstock, increasing seed production, and reducing the operational constraints associated with physiological exhaustion during extended breeding cycles.Materials and methodsBroodstockAt the end of the hatchery breeding season, Whiteleg shrimp broodstock (L. vannamei; synonym: Penaeus vannamei) sourced from Kona Bay, USA, were selected to ensure comparable baseline reproductive performance among experimental groups. Selection was based on previous spawning records and similar reproductive maturity, the latter being assessed from external signs of ovarian development visible through the dorsal exoskeleton. Male broodstock had a mean body weight of 50± 2 g and a mean total length of 17 ± 2 cm, whereas females had a mean body weight of 69 ± 5 g and a mean total length of 19 ± 2 cm. The experiment was initiated at the end of the breeding season to evaluate whether different feeding strategies could prolong the reproductive performance of the broodstock beyond the normal breeding period.SamplingBefore tissue sampling, shrimp were anesthetized by immersion in a clove-oil solution at 450 mg/L until spontaneous movement ceased. The shrimp were subsequently transferred to an ice slurry to maintain insensibility, and lymph was collected prior to dissection. Terminal dissection and tissue collection were then performed immediately while the animals remained insensible, and the collected tissues were promptly processed for the subsequent analyses. The samples were collected at the beginning of the experiment (n = 3 per treatment) and at the end of the experiment (day 30; three per replicate, n = 9 per group).Initial measurementsAt the beginning of the study, initial samples were collected to establish baseline measurements for comparison with the final experimental results and to evaluate the effects of the dietary treatments on broodstock performance and reproductive outcomes. The broodstock used for the initial assessment were collected after completion of the final hatchery spawning cycle; therefore, the females were considered to be in a post-spawning condition. The body length and weight of each shrimp were recorded, and hemolymph samples were collected into citrate–EDTA anticoagulant solution for biochemical analysis. Hepatopancreas samples were collected from both males and females, while testes from males and ovaries from females were obtained for histological examination. In addition, eyestalks and hepatopancreas tissues from females and androgenic glands from males were collected, immediately preserved in liquid nitrogen, and subsequently used for RNA extraction.These analyses were conducted to assess the health status and overall growth of the broodstock. As well as, the measurements provided critical baseline data, reflecting both the condition of the stock at the end of the breeding season and the initial state of the broodstock used in the study. This information is essential for understanding the metabolic and health status of the shrimp under the experimental conditions.Experimental setupFollowing the initial investigation, shrimp were divided into four groups, each with three replicates. Each group consisted of 18 females and 18 males.The experiment was conducted in black concrete tanks measuring 6.3 × 6.3 × 1.2 m (length × width × height). Each tank had a central drainage outlet and separate seawater and freshwater inlets, allowing the salinity to be maintained at 30 ppt. Each tank was divided into three fully separated sections using impermeable partitions, resulting in three independent experimental units, each measuring 2.0 × 6.3 m. These units served as replicates for the respective treatment groups. Water was supplied independently to each experimental unit, thereby preventing water exchange among compartments. Before the experiment began, all tanks and equipment were disinfected with formalin at a concentration of 3 ppm.After adjusting the salinity, the water underwent a series of purification and sterilization processes to ensure high quality. This included filtration through a sand filter and a protein skimmer to remove organic compounds, followed by ozone treatment and UV sterilization, and finally passing through a cotton filter. Additionally, heaters and aerators were installed on all sides to maintain proper aeration, keeping the water temperature at 29 °C.Every day at 1:00 am, the tanks were cleaned and the water was changed completely by 100%. Light cycle was at 12 h of light and 12 h of darkness.Water quality was monitored periodically, with the following parameters recorded: salinity (30 ± 0.00 ppt), temperature (29 °C), pH (8.14 ± 0.02), ammonia nitrogen (0.12 ± 0.02 mg/L), nitrite (0.02 ± 0.01 mg/L), and total alkalinity (110 ± 0.00 mg/L).Weekly water samples were collected. Isolation and enumeration of Vibrio spp. were performed using thiosulfate–citrate–bile salts–sucrose (TCBS) agar. Microbial analyses included assessment of yellow-, green-, fluorescent-, and black colony-forming Vibrio species. These analyses were conducted to evaluate water quality and monitor shrimp health, ensuring optimal rearing conditions throughout the experiment.Feeding protocolsThe experimental design followed standard feeding practices commonly employed in shrimp hatcheries. During the acclimation period, the basal feeding ration was set at approximately 25% of the shrimp biomass, consisting of 80% worms (Nereis spp., ragworms) distributed over three daily meals and 20% squid (Loligo forbesii) provided in a single meal. This feeding regime is consistent with practices commonly reported for shrimp broodstock management in commercial hatcheries42,43,44. Throughout the experimental period, all feed offered was completely consumed by the shrimp.Four feeding treatments were evaluated: Group 1 (control): 80% worms and 20% squid (calamari); Group 2: 80% worms and 20% mussels (Mytilus galloprovincialis); Group 3: 50% worms, 25% mussels, and 25% squid (calamari); and Group 4: 25% worms, 25% mussels, 25% squid (calamari), and 25% chicken liver. All ingredient proportions were calculated on a wet-weight basis. The experimental feeding period lasted 30 days.Fresh squid was cleaned by removing the entrails and eyes and then cut into approximately 1 cm pieces. All feed ingredients were sterilized using povidone-iodine prior to use. The feed components were stored at − 20 °C until feeding, except for the worms, which were offered live.Composite analysis of feed and fatty acid analysis of feed sourcesFeed samples from each ingredient were chemically analyzed for moisture, protein, fat, and ash content using standard methods established by AOAC45. The crude protein was determined using Kjeldahl method, while the crude fat was measured by the Soxhlet extraction. Moisture content was estimated by calculating the weight loss observed after heating samples in an oven at 60 °C for two days until a constant weight was achieved. The ash contents were determined by analyzing the remaining content after heating samples in a muffle furnace at 550 °C for two hours.The fatty acid composition of the feed sources was determined based on data reported in a concurrent companion study (currently unpublished; Supplementary Table S1). Briefly, fat was extracted from each feed sample and subjected to fatty acid methyl ester (FAME) preparation following the method described by Danish and Nizami46.MatingOnce feeding stabilized on the basal diets—approximately two weeks into acclimation—the experimental diets were administered at full rations for 30 days. Upon observing signs of gonadal maturation in all treatments, we proceeded with the mating process and its preparations.To ensure that only fully mature females were introduced into the male tank for mating, ovarian development was assessed visually through the dorsal exoskeleton. Females were considered ready for mating when the ovary was markedly enlarged and turgid, extended from the cephalothoracic region along the dorsal abdomen, exhibited well-developed anterior and lateral lobes, and showed a distinct dark-orange to orange-brown coloration. These features corresponded to Stage 4, indicating full ovarian maturation and advanced yolk deposition. Females meeting these criteria were transferred to the male tank at 10:00 a.m., and the number introduced was recorded for accurate tracking.The male tank is continuously observed throughout the day to record key mating behaviors, such as circular swimming patterns47. At 7:00 PM, females are carefully examined for evidence of mating success. If sperm deposition in the seminal receptacle is confirmed, mated females are promptly transferred to the hatchery tank for egg incubation, as rapid transfer enhances fertilization success.Females that do not mate are returned to their original tanks to continue their reproductive cycle, allowing them to re-enter mating conditions without disruption. This meticulous monitoring process ensures that only fertilized females are moved to the hatchery.These steps were repeated six times for each group, involving six separate matings per treatment, all conducted on the same day. This design enabled a comprehensive assessment of each group’s impact on mating behavior and reproductive success. By repeating the procedures, random variation was minimized, enhancing result accuracy and improving the reliability of the study’s data.Reproductive performance assessmentThe hepatosomatic index (HSI) and gonadosomatic index (GSI) in females were calculated using the following formulas:$$:HSI=left(frac{text{Hepatopancreas}text{:weight}}{text{Total:body:weight}}right)times:100$$$$:GSI=left(frac{text{Gonad:weight}}{text{Total:body:weight}}right)times:100$$Additional reproductive parameters were evaluated separately for each experimental replicate. Females ready for mating were identified based on the external signs of advanced ovarian maturation described above and were recorded as the number of females reaching the appropriate mating stage before pairing. Fertilized females were defined as those that spawned eggs with the presence of an externally attached spermatophore on the female thelycum. Fertilization rate was calculated as follows: fertilization rate (%) = (number of fertilized females/number of females ready for mating) × 100.Egg production was estimated using a volumetric counting method. After spawning, the eggs were uniformly distributed within a known total volume of seawater, and three subsamples of known volume were collected and counted using a graduated glass counting vessel. The mean number of eggs per unit volume was then extrapolated to the total seawater volume to estimate the total number of eggs produced in each replicate. Hatched larvae were quantified at the nauplius stage using the same volumetric counting procedure. Egg production per female was calculated by dividing the total number of eggs by the number of spawning females, while larval production per female was calculated by dividing the total number of larvae by the number of spawning females. Total egg and larval production and their corresponding per-female values were expressed as ×10⁶.In addition, the ovarian maturation stages of female shrimp were recorded daily for 30 days across four dietary treatments to assess the duration of ovarian maturation at each stage. Ovarian-stage progression was evaluated by visually examining the ovaries through the dorsal exoskeleton. Females were classified into five developmental stages, numbered 0–4, based on ovarian visibility, size, coloration, and development of the anterior and lateral lobes. The staging system was adapted from previously published macroscopic ovarian maturity criteria for L. vannamei48,49, which have been validated against histological observations49. Stage 0 represented the absence of visible ovarian development. At Stage 1, the ovary appeared as a very thin, whitish to translucent line along the dorsal region and could be clearly distinguished from the digestive tract. Stage 2 was characterized by an increase in ovarian size and a change in coloration from whitish-translucent to pale yellow. At Stage 3, the ovary became more prominent and enlarged, with a distinct orange coloration and clearly developing anterior and lateral lobes, indicating advanced ovarian development and increased yolk accumulation. Stage 4 represented full ovarian maturation, with a markedly enlarged ovary extending from the cephalothoracic region along the dorsal abdomen, well-developed anterior and lateral lobes, and a dark-orange to orange-brown coloration, indicating advanced yolk deposition and readiness for spawning. The number of females assigned to each stage was recorded for each treatment, and ovarian-stage distribution was expressed as the percentage of examined females in each stage.Hemolymph biochemical parameters and hepatopancreatic oxidative stress biomarkersEstradiol (E2) concentrations were measured using a Siemens IMMULITE 2000/XPi immunoassay analyzer (Siemens Co., Germany). Cholesterol, low-density lipoprotein cholesterol (LDL), and triglyceride concentrations were determined colorimetrically using commercial assay kits (Biodiagnostic Co., Egypt) at wavelengths of 500, 550, and 505 nm, respectively. vLDL was estimated using the Friedewald equation: vLDL = triglycerides/550.Hepatopancreatic antioxidant and oxidative-stress indicators, including superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), and catalase (CAT) levels, were determined colorimetrically using commercial assay kits (Biodiagnostic Co., Egypt) at wavelengths of 560, 340, 534, and 510 nm, respectively. MDA was measured as an indicator of lipid peroxidation.Gene expression analysisRNA was extracted from eyestalks and hepatopancreas of females, as well as androgenic glands of males, using the TRIzol reagent (Invitrogen™ Life Technologies, US). The quantity and quality of the RNA were assessed using a NanoPhotometer® NP80 (Implen, Germany) and 1% agarose gel electrophoresis.cDNA was synthesized from 2 µg of RNA using a cDNA synthesis kit (Applied Biotechnology Co., Egypt). Real-time PCR was then performed using an Applied Biosystems 7900HT real-time PCR instrument with qPCR GreenMaster (Jena Bioscience, Germany). The cycling conditions were as follows: initial denaturation at 94 °C for 2 min, followed by 40 cycles of 94 °C for 30 s (denaturation), a gene-specific annealing temperature for 30 s, and 72 °C for 30 s (extension). A dissociation curve analysis was conducted to assess the specificity of the PCR products. The primers used are listed in Table 1.Table 1 Primers used for real-time PCR.Full size tableThe expression of vitellogenesis-inhibiting hormone (vih) was analyzed in the eyestalks, while vitellogenin (vtg) was examined in the hepatopancreas of females. In males, the expression of insulin-like androgenic gland hormone (iag) and insulin-like growth factor-binding protein-related protein 1 (igfbp1) was assessed in the androgenic glands. Gene expression levels were normalized to elongation factor 1-alpha (ef1α), which was used as the internal reference gene. Because no treatment group was designated as a calibrator, normalized relative gene expression was calculated as 2−ΔCt, where ΔCt = Ct(target gene) − Ct(reference gene). Higher 2−ΔCt values indicate greater normalized transcript abundance34.Histological examinationThe gonads and hepatopancreas of adult male and female shrimp were fixed in Davidson’s solution for 24 h, then dehydrated in a graded ethanol series (70%, 80%, 90%, and 100%), cleared in xylene, embedded in paraffin, and sectioned at a thickness of 4 μm. The tissue slides were stained with hematoxylin and eosin (H&E) and examined under a light microscope (OLYMPUS CKX53, Germany) equipped with a camera (OLYMPUS EP50, Germany) for histological evaluation. Images were captured for documentation53,54.Statistical analysisData were analyzed using GraphPad Prism software (version 4.0; GraphPad Software, San Diego, CA, USA; https://www.graphpad.com/). For each measured parameter, the dietary treatment was considered the independent factor, and differences among the four treatment groups were evaluated using one-way analysis of variance (ANOVA). The assumptions of normality and homogeneity of variance were assessed before analysis using the Shapiro–Wilk test and Levene’s test, respectively. When the overall ANOVA indicated a significant treatment effect, Tukey’s honestly significant difference multiple-comparison test was used to perform all pairwise comparisons among treatment means while controlling the family-wise Type I error rate. Results are presented as mean ± standard deviation (SD), with statistical significance defined as p < 0.05.In addition, ovarian maturation stages of female shrimp were recorded daily for 30 days across four dietary treatments (Groups 1–4). Each individual was classified into one of five ordinal developmental stages (Stages 0–4). Because maturation stage represents an ordered categorical variable, the effects of diet and time were analyzed using ordinal logistic regression (cumulative link model)55. The model was defined as:$$:logitleft[Pleft(Yge:jright)right]={alpha:}_{j}+{beta:}_{1}:left(Groupright)+{beta:}_{2}:left(Dayright)$$where Y represents the ovarian stage and αj denotes the threshold intercepts between stages. Counts of females at each stage were used as frequency weights56.To quantify overall ovarian development, an ovary maturity index (OMI) was calculated as the weighted mean maturity stage based on the distribution of females across stages, an approach conceptually similar to weighted maturity indices derived from the distribution of gonadal developmental stages57,58. Such indices provide a quantitative representation of reproductive development and complement traditional measures such as the gonadosomatic index59,60. The OMI was calculated as follows:$$:text{O}text{M}text{I}:=:frac{sum:(itimes{n}_{i})}{sum:{n}_{i}}$$where i represents the maturation stage (0–4) and ni is the number of females at that stage. These statistical analyses were performed using the statsmodels ordinal package in Python61, with significance set at α = 0.05.ResultsProximate composition and fatty acid profile of the dietsThe formulation in Group 4, consisting of equal parts (25%) worms, mussels, squid (calamari), and chicken liver, had the highest protein percentage, followed by Group 3, which contained 50% worms, 25% mussels, and 25% squid (calamari) (Table 2). The lowest protein percentage was found in Group 2, which included 80% worms and 20% mussels. Group 4 also had the highest crude fat percentage, while the other formulations showed similar levels. Ash content was highest in Group 2, followed by Group 1, Group 3, and Group 4 (Table 2).Table 2 Composite analysis of different diet formulations using worms, squid (calamari), mussels, and chicken liver.Full size tableThe diet formulation used in this study was identical to that described in a concurrent (unpublished) study; the complete fatty acid profile is provided in Supplementary Table S1 for reference. The fatty acid composition of the experimental diets varied significantly, reflecting differences in the proportions of marine and terrestrial ingredients.Group 1, which served as the control diet dominated by polychaetes, was characterized by higher levels of elaidic acid (approximately 1.2% higher than in Group 3), along with notable concentrations of heptadecanoic acid and cis-11,14,17-eicosatrienoic acid. Similar to Group 2, its profile reflects a traditional worm-based diet that, although rich in certain marine lipids, lacked the diversity observed in the higher-performing treatments. Group 2 contained the highest concentrations of palmitic acid, palmitoleic acid, and cis-5,8,11,14,17-eicosapentaenoic acid (EPA).In contrast, the more diversified diets—particularly Group 4—were distinguished by significantly higher levels of linoleic acid (ω−6), oleic acid, and cis-13,16-docosadienoic acid. Additionally, erucic acid, tetracosanoic acid, arachidic acid and cis-11-eicosenoic acid were markedly elevated in both Groups 3 and 4 compared with the worm-dominated diets (Groups 1 and 2). The diets of Groups 2 and 3 contained approximately 1% higher levels of α-linolenic acid (ω−3) than those of Groups 1 and 4. In contrast, the diet of Group 1 contained substantially lower levels of cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) compared with the diets of the other groups.Fatty acids such as butyric, hexanoic, dodecanoic, and behenic acids were present only at negligible levels across all formulations.Female fecundityReproductive performance of shrimp females varied among the four dietary treatments (Group 1–Group 4) (Table 3). Diet significantly affected both the hepatosomatic index (HSI) and gonadosomatic index (GSI), with the highest values recorded in Group 3.Table 3 Female fecundity parameters of female shrimp fed four different diets.Full size tableDiet also significantly influenced the number of females available for reproduction (p = 0.043). Similarly, the number of fertilized females differed significantly among treatments (p = 0.019), with the highest values observed in Group 3 (3.00 ± 0.63) and Group 4 (2.83 ± 1.17). In contrast, fertilization rate did not differ significantly among dietary treatments (p = 0.062).Egg production varied markedly among treatments. Females fed the Group 3 diet produced the highest number of eggs, followed by Group 4, whereas the lowest egg production was observed in Group 2. Statistical analysis confirmed a significant effect of diet on total egg production (p = 0.013). Likewise, the number of eggs per female differed significantly among groups (p = 0.015). Group 3 exhibited a higher eggs-per-female ratio than Group 2, while Groups 1 and 4 showed intermediate values that did not differ significantly from the other treatments.Larval production followed a similar pattern. The highest larval output was recorded in Group 3 (10.34 ± 7.20 × 10⁶ larvae), which was significantly higher than that observed in Groups 1 and 2 (Table 3). Diet also had a significant effect on larval production (p = 0.0077). In addition, the larvae-per-female ratio varied significantly among treatments (p = 0.0015). Group 3 produced the highest number of larvae per female, significantly exceeding the values recorded for Groups 1 and 2. Larval production in Group 4 was intermediate and did not differ significantly from the other treatments.Ovarian development increased significantly over the 30-day experimental period across all treatment groups (β = 0.132, p < 0.001). Dietary treatment significantly influenced the maturation trajectory and the rate at which females transitioned from immature to mature stages (Fig. 1).Fig. 1Full size imageTemporal progression of the Ovary Maturity Index (OMI) in female shrimp fed four experimental diets over a 30-day period. The OMI was calculated as the weighted average of ovarian maturation stages (0–4), with higher values indicating more advanced maturation. Data points represent the daily mean OMI for each treatment group. Groups 3 (green) and 4 (red-orange) exhibited significantly higher maturation rates compared with the Group 1 control (blue) (p < 0.001). Group 2 (orange) showed the lowest overall maturation rate during the first half of the experimental period.Group 3 exhibited the strongest overall performance throughout the trial, with a significantly higher probability of progressing to advanced maturation stages compared with the Group 1 control (β = 0.658, p < 0.001). This response was characterized by an early transition out of Stage 0, as reflected in the distribution of ovarian developmental stages (Fig. 2). Similarly, Group 4 also outperformed the control group (β = 0.503, p < 0.001), showing a marked increase in reproductive readiness during the latter half of the experiment.Fig. 2Full size imageDistribution of ovarian developmental stages in female shrimp across four dietary treatments (Groups 1–4) during a 30-day feeding trial. Stacked bars show the percentage of individuals at each developmental stage: Stage 0 (dark purple; immature), Stage 1 (blue, early development), Stage 2 (teal, vitellogenesis), Stage 3 (green, late vitellogenesis), and Stage 4 (yellow; fully mature/spawning ready). A rapid transition from immature to advanced maturation stages (Stages 3–4) is most evident in Group 3 beginning around Day 13. By Day 30, Groups 3 and 4 displayed the highest proportion of reproductively ready females compared with the control (Group 1) and Group 2.In contrast, Group 2 showed a tendency toward slower maturation during the first two weeks of the study (β = −0.187), although it was not significantly different from the Group 1 control by the end of the trial (p = 0.132). Ovary Maturity Index (OMI) values calculated at Day 20 further supported these findings, with Groups 3 and 4 reaching an index of 2.50, substantially higher than the Group 1 baseline (OMI = 1.38) at the same time point (Fig. 1).Analysis of the stage distribution indicated that by the end of the trial (Day 30), Group 4 achieved the highest final maturity index (OMI = 2.56), while Group 3 maintained the most consistent proportion of Stage 4 (fully mature) females during the final 10 days of the experiment (Fig. 2).Hemolymph biochemistry and hepatopancreatic oxidative stress biomarkersFemalesIn female shrimp, hemolymph biochemical parameters exhibited variable responses among the experimental groups (Table 4). Estradiol (E2) concentrations were markedly lower than the initial values recorded before the experimental diets were introduced. However, Group 4 showed a slight but significant increase compared with the other treatment groups. Cholesterol levels did not differ significantly among the groups or from the initial measurements, with the exception of Group 2, which demonstrated a noticeable increase. Low-density lipoprotein (LDL) concentrations were higher in Groups 2 and 3; however, they remained lower than the initial measurements. In contrast, very-low-density lipoprotein (vLDL) reached its highest level in Group 4, followed by Group 3. Triglycerides displayed a trend similar to that observed for vLDL.Table 4 Hematological parameters and hepatopancreatic oxidative stress biomarkers in female shrimp fed four different diets.Full size tableRegarding hepatopancreatic oxidative stress biomarkers, superoxide dismutase (SOD) activity was significantly higher in Group 4, while no significant differences were detected among the remaining groups or compared with the initial values (Table 4). Glutathione peroxidase (GPx) activity was highest in Groups 2 and 3 and moderate in Group 4. Malondialdehyde (MDA) levels were markedly elevated in Group 2. In contrast, catalase activity did not differ significantly among the experimental groups (Table 4).MalesIn male shrimp, hemolymph biochemical profiles showed a different pattern compared with females (Table 5). E2 levels were lowest in Group 2 and highest in Group 3, while the remaining groups did not differ significantly from each other or from the initial measurements. Cholesterol and LDL concentrations were highest in Group 4 and lowest in Group 3. In contrast, vLDL levels peaked in Group 2, showed moderate reductions in Group 4, and reached their lowest values in Group 3. Triglyceride levels differed significantly among male groups and relative to the initial measurements, with the highest values in Group 2 and the lowest in Group 3. Group 4 also showed relatively high triglyceride levels; however, these did not differ significantly from those in Group 1.Table 5 Hematological parameters and hepatopancreatic oxidative stress biomarkers in male shrimp fed four different diets.Full size tableHepatopancreatic oxidative stress biomarkers varied among the groups. SOD activity was substantially reduced in Group 2 and was moderately lower in Group 3 compared with the other groups and the initial values (Table 5). Similarly, GPx activity was reduced in Groups 2 and 3 relative to the other treatments. MDA levels did not show marked variation among the male groups, although the lowest values were recorded in Group 1. In contrast, catalase activity increased in Groups 2 and 3 and reached its highest level in Group 4 (Table 5).Expression patterns of reproduction-related genesFemalesIn females, the expression of vitellogenesis-inhibiting hormone (vih) in the eyestalks showed a gradual downregulation from Group 1 to Group 4 compared with the initial stage prior to the introduction of the experimental diets (Fig. 3; p < 0.0001; Supplementary File 2). In contrast, vitellogenin (vtg) expression in the hepatopancreas increased progressively from Group 2 to Group 4, with no significant difference observed between the initial stage and Group 1 (Fig. 3; p < 0.0001; Supplementary File 2).Fig. 3Full size imageExpression of vitellogenesis-inhibiting hormone (vih) in the eyestalks and vitellogenin (vtg) in the hepatopancreas of female broodstock shrimp (L. vannamei) fed different experimental diets. Gene expression levels were normalized to elongation factor 1-alpha (ef1α) as the internal reference gene. Results are presented as mean ± SD (n = 9 per group). Relative expression was calculated using the ΔCt method and expressed as 2−ΔCt for clarity. The overall ANOVA indicated a significant effect of dietary treatment (P < 0.0001). Tukey-adjusted P values for the pairwise comparisons are shown between groups.MalesIn the androgenic glands, the expression levels of insulin-like androgenic gland hormone (iag) and insulin-like growth factor-binding protein–related protein 1 (igfbp1) showed a significant increase from Group 1 to Group 4 compared with the initial measurements. However, no significant difference was observed between Groups 3 and 4 (Fig. 4; Supplementary File 2).Fig. 4Full size imageExpression of insulin-like androgenic gland hormone (iag) and insulin-like growth factor-binding protein-related protein 1 (igfbp1) in the androgenic glands of male broodstock shrimp (L. vannamei) fed different experimental diets. Gene expression levels were normalized to elongation factor 1-alpha (ef1α) as the internal reference gene. Results are presented as mean ± SD (n = 9 per group). Relative expression was calculated using the ΔCt method and expressed as 2−ΔCt for clarity. The overall ANOVA indicated a significant effect of dietary treatment (P < 0.0001). Tukey-adjusted P values for the pairwise comparisons are shown between groups.Histological results of hepatopancreas, ovaries and vas deferensHepatopancreasAt the initial stage, the hepatopancreas showed preserved tubular architecture with distinct lumina and identifiable B, R, and F cells. B cells contained a prominent cytoplasmic vacuole, R cells displayed multiple smaller vacuoles, and F cells appeared comparatively basophilic and less vacuolated. No obvious epithelial disruption or cellular debris was observed within the tubule lumina (Fig. 5A).Fig. 5Full size imagePhotomicrographs of histological cross-sections of the hepatopancreas from experimental adult L. vannamei females (H&E staining). L, lumen of hepatopancreatic tubules; B, B-cells; F, F-cells; R, R-cells; E, E-cells.In Group 1, the hepatopancreatic epithelium exhibited prominent cytoplasmic vacuolation, particularly in regions containing R and B cells, together with irregular variation in lumen width (Fig. 5B). Group 2 showed generally rounded or oval tubules with distinct, frequently star-shaped lumina and numerous epithelial vacuoles. No clear epithelial necrosis or cellular sloughing was evident (Fig. 5C).Group 3 exhibited extensive epithelial vacuolation, variable epithelial thickness, and irregular tubule-lumen morphology, although overt epithelial destruction was not apparent (Fig. 5D). In Group 4, the tubules displayed well-defined star-shaped lumina surrounded by a relatively compact epithelial lining. Cytoplasmic vacuolation appeared less extensive than in Groups 1 and 3, and the overall tubular architecture was comparatively well preserved (Fig. 5E).In males at the initial stage, the hepatopancreas showed generally preserved tubule architecture with distinct lumina and identifiable B, F, and R cells. The epithelial cells displayed prominent cytoplasmic vacuolation, while the epithelial lining remained generally continuous, with no obvious cellular debris or extensive epithelial disruption within the lumina (Fig. 6A).Fig. 6Full size imagePhotomicrographs of histological cross-sections of the hepatopancreas from experimental adult L. vannamei males (H&E staining). L, lumen of hepatopancreatic tubules; B, B-cells; F, F-cells; R, R-cells; E, E-cells.In Group 1, the hepatopancreatic tubules retained their general organization and continuous epithelial lining. Prominent cytoplasmic vacuolation was observed in the epithelial cells, together with variation in lumen shape and width. However, an increase in vacuolation relative to the initial stage could not be established from the representative sections alone (Fig. 6B). Group 2 exhibited generally preserved tubule profiles with distinct, frequently irregular or star-shaped lumina. Numerous cytoplasmic vacuoles of variable size were present within the epithelium, but no clear evidence of extensive epithelial disruption or cellular sloughing was observed (Fig. 6C).In Group 3, the hepatopancreas showed heterogeneous tubule morphology, with prominent epithelial vacuolation and variation in lumen size and shape. Several tubules exhibited enlarged or irregular lumina, although definite epithelial thinning could not be confirmed consistently throughout the section (Fig. 6D). In Group 4, the tubules displayed relatively well-defined, frequently star-shaped lumina surrounded by a generally continuous epithelial lining. Cytoplasmic vacuolation remained evident, while the overall tubular architecture appeared comparatively preserved. A definite reduction in vacuolation relative to the other groups would require quantitative histomorphometric assessment (Fig. 6E).OvariesAt the initial stage, the ovarian tissue was composed predominantly of small primary-growth or previtellogenic oocytes, with some larger developing oocytes. The oocytes exhibited distinct nuclei and were supported by ovarian stroma containing visible blood vessels. No overt tissue disruption or degenerative changes were evident in the examined fields (Fig. 7A, B).Fig. 7Full size imagePhotomicrographs of histological cross-sections of ovaries from experimental L. vannamei females, showing multiple stages of oocyte maturation (H&E staining). P, perinucleolar stage; Pg, primary growth ovarian follicle; Lc, lipid and cortical alveoli stage; Vf, vitellogenic follicle; Ev, early vitellogenic stage; Lv, late vitellogenic stage; Pv, post-vitellogenic stage; Ca, cortical alveoli; post-ovulatory follicle (asterisk); yolk granules (arrowhead); N, nucleolus.In Group 1, the ovary remained dominated by small primary-growth and previtellogenic oocytes, together with a limited number of larger developing oocytes. Overall, the ovarian organization was generally preserved, but clear advancement of oocyte maturation relative to the initial stage was not evident (Fig. 7C, D). Group 2 displayed a greater occurrence of enlarged, vitellogenic oocytes with more abundant eosinophilic cytoplasm and distinct nuclei, while smaller primary-growth oocytes remained present. These features indicate progression of ovarian development relative to the initial stage and Group 1 (Fig. 7E, F).In Group 3, numerous large vitellogenic oocytes containing abundant yolk material were observed. The presence of prominent nuclei and cortical structures was consistent with a comparatively advanced stage of oocyte development (Fig. 7G, H). Group 4 contained a mixture of primary-growth, vitellogenic, and advanced or postvitellogenic oocytes, indicating continued ovarian maturation (Fig. 7I, J).vas deferensAt the initial stage, the vas deferens exhibits a healthy and fully functional organization, characterized by clear, unobstructed lumina, a delicate reticular framework, and well-defined basement membrane, submucosa, epithelial, and muscle layers. Cellular density is moderate, with evenly distributed nuclei. The epithelium consists of columnar cells with eosinophilic cytoplasm and prominent apical secretory material or brush border (arrowhead), indicating normal reproductive activity. The lumen is well defined and contains a dense sperm mass (Fig. 8A, B).Fig. 8Full size imagePhotomicrographs of histological cross-sections of the vas deferens from experimental adult L. vannamei males (H&E staining). Vd, vas deferens; SM, sperm mass; EPT, epithelial lining; MS, muscle layer.In Group 1, relative to the initial stage, the vas deferens shows slightly dilated lumina, subtle thickening of the submucosa, and minor architectural disturbances. Overall tissue organization remains largely preserved; however, the epithelial layer appears mildly reduced in height compared with the initial stage (Fig. 8C). The sperm mass remains compact. Fewer apical secretory granules (arrowhead) and a slightly thinner epithelium are evident (Fig. 8D). In Group 2, more pronounced alterations are observed compared with the initial stage. The lumina are well defined but variable in size, and cellular density is increased, with densely packed nuclei suggestive of epithelial hyperplasia. Epithelial cells display heightened secretory activity and irregular mucosal folding, indicating a proliferative response. Intense apical secretory material is evident (arrowhead; Fig. 8E, F).In Group 3, the vas deferens exhibits slightly constricted lumina, a reduction in sperm mass volume, and altered mucosal folding (Fig. 8G). Focal epithelial cell vacuolization is observed (arrowhead), and although the muscle layer remains present, overall mucosal integrity appears compromised (Fig. 8H). In Group 4, the overall architecture shows a moderately tall epithelium with abundant apical secretory material, resembling Group 2 but to a lesser extent. Columnar epithelial cells retain visible secretory granules (arrowhead; Fig. 8I, J). The sperm mass is present but appears less dense than that observed at the initial stage.DiscussionThe reproductive success of L. vannamei (whiteleg shrimp) is closely linked to broodstock nutrition because the diet provides the biochemical precursors required for vitellogenesis and spermatogenesis. In the present study, diversified feeding strategies were evaluated as a means of extending reproductive capacity toward the end of the commercial breeding season, a period typically characterized by physiological exhaustion and declining fecundity.The results demonstrate that dietary composition, particularly the inclusion and relative proportions of worms, mussels, squid, and chicken liver, substantially influenced reproductive performance. Females fed the Group 3 diet, consisting of 50% worms, 25% mussels, and 25% squid, exhibited the highest fecundity, egg production, larval output, and rate of ovarian maturation. The Group 4 diet, containing equal proportions of worms, mussels, squid, and chicken liver, also significantly improved reproductive readiness and maturity indices compared with the control and Group 2 diets. Proximate composition analysis further showed that Group 4 had the highest overall protein and lipid contents, indicating that nutrient-dense, diversified diets may help meet the considerable energetic and nutritional demands of gametogenesis.Collectively, these findings indicate that balanced combinations of marine-derived protein sources, with complementary lipid and fatty-acid profiles, can synergistically enhance reproductive performance, endocrine activity, and tissue integrity in broodstock, consistent with previous reports62,63. Although polychaetes (worms) are traditionally favored due to their high levels of highly unsaturated fatty acids (HUFAs)21,22,64,65, worm-dominated diets alone (Groups 1 and 2) did not yield optimal outcomes during the late breeding season. Instead, incorporating additional marine ingredients such as mussels and squid appears to improve the balance of essential fatty acids—particularly omega-3 to omega-6 ratios and levels of EPA and DHA—while also supplying key micronutrients, including sterols and phospholipids, that support vitellogenesis and ovarian maturation63. The superior performance of Group 3 suggests that maintaining a relatively high proportion of worms, while complementing them with other marine protein sources, optimizes reproductive output without introducing potential imbalances associated with terrestrial ingredients such as chicken liver, which is comparatively rich in polyunsaturated fatty acids and minerals28. Overall, these results support the concept that diversified broodstock diets can prolong reproductive efficiency and sustain spawning performance toward the end of the production cycle, in line with earlier findings in penaeid shrimp16,66,67.Interestingly, Group 4 exhibited significantly higher levels of linoleic and oleic acids. Beyond serving as energy sources, these fatty acids act as precursors for eicosanoid synthesis68,69,70, which regulates key reproductive processes in crustaceans. Notably, the elevated EPA levels observed in Group 2 did not translate into superior fecundity, underscoring that a balanced “cocktail” of lipid classes and amino acids—such as that provided by diversified protein sources—is more effective than a high concentration of any single fatty acid24,40.Proximate and fatty-acid analyses confirmed substantial variation among diets in palmitic, palmitoleic, linoleic, α-linolenic, EPA, and DHA contents, which likely underpin the observed reproductive differences. In penaeids, vitellogenesis is highly sensitive to lipid quality: long-chain polyunsaturated fatty acids (LC-PUFAs), phospholipids, and sterols are essential for oocyte membrane formation, yolk lipoprotein synthesis, and embryonic development13,71. Dietary provision of EPA and DHA has been consistently linked to improved egg quality and larval survival24,27. The combined presence of high-quality protein and a favorable fatty-acid profile in Groups 3 and 4 likely enhanced vitellogenin synthesis in the hepatopancreas and its transport to developing oocytes.The hepatosomatic index (HSI) and gonadosomatic index (GSI) are reliable indicators of nutritional status and reproductive maturation15,59. GSI increases progressively with ovarian development, and both indices are closely linked during vitellogenesis15,59. The peak HSI and GSI values observed in Group 3 indicate efficient mobilization of nutrients from the hepatopancreas to the ovaries, consistent with the elevated egg production and larval output recorded in this group.In line with these observations, the ovary maturity index (OMI) and ordinal logistic regression provided a high-resolution temporal profile of maturation. Groups 3 and 4 showed a rapid transition from immature (Stage 0) to advanced vitellogenic stages (Stages 3 and 4) by the second week of the trial. By Day 20, both groups reached an OMI of 2.50, significantly exceeding the control (OMI = 1.38). These findings suggest that diversified marine protein sources enhance nutrient availability for vitellogenesis and ovarian maturation, helping to overcome reproductive stagnation in aging broodstock and extend the effective spawning window14,72,73.Molecular responses provided additional insight into the dietary effects on female reproduction. Normalized vih transcript abundance in the eyestalk decreased from Group 2 to Group 4, whereas hepatopancreatic vtg abundance showed the opposite pattern. This inverse association is consistent with the established Vih–Vtg regulatory axis in L. vannamei, in which Vih produced by the eyestalk and brain suppresses hepatopancreatic vitellogenin synthesis17,59. Nutritional signals can modulate this axis through energy status, lipid-derived mediators, and nutrient-sensing pathways (e.g., insulin-like signaling), thereby relieving inhibition and promoting vitellogenesis63. Therefore, the lower vih and higher vtg transcript abundance in Groups 3 and 4 suggests reduced inhibitory signaling and greater capacity for yolk-precursor synthesis. Nutrient availability may also regulate vitellogenesis through metabolic signaling pathways72.The treatment comparisons indicate that molecular activation did not translate linearly into reproductive output. Group 2, which received worms and mussels, exhibited the lowest egg and larval production, consistent with its comparatively weaker vitellogenic response. Group 1, which received worms and squid, produced intermediate reproductive results. Group 3, which combined worms, mussels, and squid, achieved the highest GSI, number of fertilized females, and egg and larval production, suggesting that this combination provided the most effective nutritional balance for ovarian maturation and subsequent reproductive performance. Although Group 4 showed a strong molecular response, including lower vih and higher vtg transcript abundance, its reproductive outputs remained below those of Group 3. This discrepancy indicates that increased vtg transcript abundance alone does not ensure maximum fecundity and that nutrient balance, ovarian developmental stage, spawning timing, and post-transcriptional regulation may also influence reproductive performance.This response is likely mediated by diet-induced stimulation of the neuroendocrine system. For example, increased estradiol (E2) levels in Group 4 females coincided with higher vtg expression, consistent with the known role of E2 in promoting yolk protein synthesis in the hepatopancreas63,74,75. The ability of the Group 4 diet to modulate this axis late in the breeding season highlights its potential as a functional broodstock feed, rather than merely a source of energy.Male reproductive performance, although often overlooked, is equally dependent on nutrition. The androgenic gland regulates spermatogenesis and male differentiation through the secretion of insulin-like androgenic gland hormone (iag), which interacts with binding proteins such as igfbp18,76. The observed upregulation of iag and igfbp1 from Group 1 to Group 4 indicates that diet also enhances male reproductive physiology. Elevated iag expression is associated with increased spermatogenic activity, androgenic gland function, and the development of secondary sexual characteristics39. The higher expression levels in Groups 3 and 4 therefore suggest improved male condition and potentially enhanced mating success or sperm quality, contributing to the increased fertilization rates and larval outputs observed. The absence of significant differences between these two groups in male gene expression further suggests that, once a nutritional threshold is reached, activation of male reproductive pathways may plateau.Elevated iag expression in the dietary groups suggests that dietary composition influenced androgenic-gland activity at the transcriptional level. Given the established role of IAG in male sexual differentiation and reproductive regulation in crustaceans, this response may reflect modulation of male reproductive physiology77,78. This molecular response was accompanied by generally preserved vas deferens architecture across the dietary groups, including Groups 2 and 4, with continuous epithelial linings, visible apical epithelial material, and intraluminal sperm masses. These features suggest maintenance of vas deferens structure and epithelial function. The inclusion of chicken liver in the Group 4 diet—providing cholesterol, fat-soluble vitamins, and other nutrients—may have supplied substrates supporting androgenic gland hormone production and protein synthesis28,79.Hemolymph lipid indicators (cholesterol, LDL, vLDL, and triglycerides) varied with both diet and sex, providing insight into metabolic partitioning during reproductive preparation. Very-low-density lipoprotein (vLDL) plays a central role in yolk protein assembly and lipid transport to the ovaries in decapods29,80. Lipids more broadly are critical to reproductive success: triglycerides support molting and early embryonic development; phospholipids influence spawning frequency; EPA and DHA contribute to fecundity and hatching success; and total HUFAs are linked to improved hatch rates37. Dietary cholesterol has also been shown to stimulate ovarian development and vitellogenin production in L. vannamei23,67, as well as increase mating rates23. Similarly, supplementation with krill oil (rich in EPA and DHA) and phospholipids modulates genes involved in cholesterol metabolism, arachidonic acid pathways, and ovarian steroidogenesis16,48.In this context, the elevated vLDL and triglyceride levels observed in Group 4 females are consistent with active lipid mobilization for oogenesis. However, Group 3 achieved superior egg and larval production despite slightly lower vLDL levels, suggesting more efficient lipid utilization and vitellogenesis rather than simple lipid accumulation. Differences in lipid profiles between males and females further underscore sex-specific allocation strategies, with females prioritizing gonadal development and males allocating more resources toward somatic maintenance81,82.Reproduction imposes substantial metabolic demands that can lead to oxidative stress through the accumulation of reactive oxygen species (ROS)35,83. Antioxidant enzyme activities (SOD, GPx, and catalase) and lipid peroxidation levels (MDA) revealed diet-dependent patterns in the hepatopancreas. In females, Group 4 exhibited significantly elevated SOD activity, while GPx peaked in Groups 2 and 3. Notably, MDA—an indicator of lipid peroxidation—was highest in Group 2. Increased antioxidant activity in broodstock likely reflects heightened metabolic rates and the associated risk of oxidative damage during vitellogenesis84,85. Group 3, which yielded the highest larval output, maintained moderate GPx activity alongside lower MDA levels than Group 2, indicating reduced oxidative damage coupled with effective reproductive investment. In contrast, the elevated MDA in Group 2 suggests increased lipid peroxidation, which may compromise oocyte quality and help explain its lower reproductive performance despite certain favorable fatty-acid characteristics32. In males, reduced SOD and GPx activities in Groups 2 and 3 may reflect shifts in metabolic allocation toward gametogenesis86.Maintaining oxidative balance is therefore essential to protect lipid-rich yolk reserves from peroxidative damage. Diets that combine beneficial fatty acids with adequate antioxidant components—such as tocopherols and selenium—are likely to enhance broodstock performance and egg quality36,87. The diversified ingredients in Group 4 likely contributed a broader spectrum of exogenous antioxidants, including selenium and vitamin E from liver and mussels25,88, thereby supporting cellular protection and physiological resilience36,89. A modest increase in catalase activity observed in Group 4 males further supports the role of this diet in maintaining physiological homeostasis during peak reproductive activity, as moderate elevations in catalase activity may confer protective benefits against oxidative stress90.Histological examination of the hepatopancreas, ovaries, and vas deferens provided morphological support for the biochemical and reproductive responses. In Group 3, the hepatopancreas showed prominent epithelial vacuolation and variation in tubule and lumen morphology, suggesting altered metabolic activity during reproductive development and sustained vitellogenin synthesis80,91. Group 4 exhibited comparatively preserved hepatopancreatic architecture, with well-defined lumina and compact epithelial organization, indicating maintenance of hepatopancreatic function during reproductive maturation92.Group 2 displayed extensive epithelial vacuolation and irregular tubule morphology, consistent with its elevated MDA levels and lower reproductive performance. These alterations may impair the digestive and biosynthetic functions required for vitellogenesis and ovarian development93,94. Ovarian histology showed advanced oocyte development in Groups 3 and 4, with numerous vitellogenic oocytes. The sustained occurrence of Stage 4 females in Group 3 was consistent with its higher egg and larval production. This reproductive pattern may be associated with the nutritional value of feed ingredients such as chicken liver, which supplies bioavailable amino acids, polyunsaturated fatty acids, vitamins, and essential minerals that may support ovarian development and reproductive metabolism28.The hepatopancreas, as the central metabolic organ in shrimp, is particularly sensitive to nutritional status. Group 1 exhibited prominent epithelial vacuolation and variation in tubule and lumen morphology—may reflect nutrient depletion typical of late-season broodstock92,95. In contrast, Group 4 showed comparatively preserved tubular architecture, with well-defined star-shaped lumina and less extensive vacuolation, more closely resembling a healthy baseline condition.Finally, vas deferens histology revealed clear diet-dependent differences in epithelial activity and sperm density. Group 2 showed epithelial hyperplasia and intense apical secretion, potentially indicative of compensatory or stress-related responses96,97, In contrast, Group 4 exhibited a more balanced secretory profile, likely supporting efficient sperm maturation and transfer, and ultimately contributing to improved reproductive success97,98,99.When considered together with the observed variation in fatty-acid composition, the proximate-composition results demonstrate that the diets differed in both nutrient quantity and lipid quality, in addition to the number and combination of ingredients. Although Groups 1–3 contained comparable amounts of crude fat (8.22–8.25%), similar total lipid concentrations do not imply nutritional equivalence because diets with comparable lipid contents may differ substantially in their proportions of saturated, monounsaturated, and polyunsaturated fatty acids, including essential fatty acids such as EPA and DHA. Such differences can influence fatty-acid digestibility, tissue fatty-acid deposition, lipid metabolism, growth, and physiological condition100,101,102,103. The variability in fatty-acid composition observed among the present treatments therefore provides additional evidence that their biological effects cannot be interpreted solely in relation to dietary diversity.The treatments also differed in crude-protein concentration. Groups 1 and 2 both contained 80% worms, but substitution of squid with mussels was accompanied by a reduction in crude protein from 60.44% to 54.62% and an increase in ash from 9.97% to 10.36%. Consequently, any biological difference between these treatments may reflect differences in protein supply, amino-acid composition, digestibility, palatability, fatty-acid profile, or mineral composition rather than the replacement ingredient alone. Previous ingredient-evaluation studies have demonstrated that animal protein sources can differ in nutrient digestibility, utilization, and growth responses even when diets are formulated to contain comparable protein and energy concentrations104.Group 3, which contained three animal ingredients, had the second-highest crude-protein concentration (64.08%) and lower ash than Groups 1 and 2, whereas Group 4, containing four ingredients, had the highest crude protein (66.74%) and crude fat (9.29%) and the lowest moisture and ash contents. Dietary protein and lipid concentrations, their ratio, and lipid source can independently or interactively affect feed intake, growth, feed efficiency, body composition, fatty-acid deposition, blood biochemical indices, antioxidant status, and nitrogen excretion101,105,106,107.The strong reproductive performance observed in Groups 3 and 4 suggests that their dietary compositions supplied complementary nutrients that supported high-quality gamete production. In many invertebrates, lipids derived from mollusks, such as mussels, and cephalopods, such as squid, contribute to the deposition of DHA and other essential phospholipids in the egg yolk, thereby supporting embryonic development and larval viability108. Nevertheless, the responses differed between the two dietary treatments. Group 3 appeared to provide a more balanced reproductive response across sexes, with particularly favorable outcomes in males, whereas Group 4 produced comparatively stronger responses in females. These findings suggest that male and female broodstock may have distinct nutritional requirements for optimal reproductive performance.Because broodstock were assessed during the late phase of the reproductive season, further studies spanning the full reproductive cycle are needed to fully elucidate the long-term effects of these feed formulations on reproductive performance in L. vannamei.ConclusionThis study demonstrates that a heavy reliance on polychaetes alone is insufficient for sustaining reproductive performance toward the end of the breeding season in L. vannamei. In contrast, diversified feeding strategies—particularly the 50:25:25 worm/mussel/squid formulation (Group 3) and the four-component diet including chicken liver (Group 4)—substantially enhance reproductive outcomes. Group 3 was most effective in maximizing egg production and larval output, while Group 4 conferred pronounced molecular and physiological advantages, including improved gene expression profiles and antioxidant capacity. Overall, implementing diversified broodstock diets offers a practical and effective strategy for hatcheries to maintain high seed production and reproductive efficiency, even as broodstock approach the later stages of the production cycle.
Data availability
The majority of the data supporting the findings of this study are included within the manuscript. Other relevant datasets can be obtained from the corresponding author upon reasonable request.
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Download referencesAuthor informationAuthors and AffiliationsGenetics and Biotechnology, Department of Aquaculture, Faculty of Fish Resources, Suez University, Suez, 43221, EgyptHaitham G. Abo-Al-ElaShrimp Hatchery, National Company for Fisheries and Aquaculture, Delta Aquaculture Sector, Kafr El-Sheikh, Ghalioun, EgyptAmira MosbahDepartment of Aquaculture, Faculty of Fish Resources, Suez University, Suez, 43221, EgyptAl-Azab TahounAuthorsHaitham G. Abo-Al-ElaView author publicationsSearch author on:PubMed Google ScholarAmira MosbahView author publicationsSearch author on:PubMed Google ScholarAl-Azab TahounView author publicationsSearch author on:PubMed Google ScholarContributionsConceptualization: Abo-Al-Ela H.G. and Tahoun A. Data Curation: Abo-Al-Ela H.G. and Mosbah A. Formal analysis: Abo-Al-Ela H.G. and Mosbah A. Investigation: Abo-Al-Ela H.G., Mosbah A., and Tahoun A. Methodology: Abo-Al-Ela H.G., Mosbah A. Writing – original draft: Abo-Al-Ela H.G. Writing – review & editing: Abo-Al-Ela H.G. All authors discussed the results and commented on the manuscript.Corresponding authorCorrespondence to
Haitham G. Abo-Al-Ela.Ethics declarations
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The authors declare no competing interests.
Declaration of Generative AI and AI-Assisted Technologies in the Writing Process
The authors used ChatGPT to improve the language and readability of this manuscript. Following its use, all authors reviewed and edited the text as necessary and accept full responsibility for the final content of the manuscript.
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The experiment was conducted in accordance with both international and national standards for animal research, having received approval from the Faculty of Fish Resources at Suez University, Egypt (Approval No. SUEZ Sci_IRB: 31/12/2025/36). The study adheres to the ARRIVE guidelines (https://arriveguidelines.org/).
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Reprints and permissionsAbout this articleCite this articleAbo-Al-Ela, H.G., Mosbah, A. & Tahoun, AA. Different dietary compositions shape reproductive physiology in Litopenaeus vannamei broodstock.
Sci Rep 16, 31483 (2026). https://doi.org/10.1038/s41598-026-71783-wDownload citationReceived: 02 July 2026Accepted: 11 September 2026Published: 08 October 2026Version of record: 08 October 2026DOI: https://doi.org/10.1038/s41598-026-71783-wShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
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KeywordsBroodstock nutritionFatty acid compositionGonadal development
Litopenaeus vannamei
Reproductive performanceVitellogenesis More
