Comparative analysis of the reproductive performance and offspring quality of pond-reared and wild-caught pharaoh cuttlefish, Sepia pharaonis broodstock

Comparative analysis of the reproductive performance and offspring quality of pond-reared and wild-caught pharaoh cuttlefish, Sepia pharaonis broodstock | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative analysis of the reproductive performance and offspring quality of pond-reared and wild-caught pharaoh cuttlefish, Sepia pharaonis broodstock Jianbo Zhang, Qilong Huang, Jiayi Sun, Jiawen Yu, Ruibing Peng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8293058/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract The Pharaoh cuttlefish （( Sepia pharaonis ） is an important species of cuttlefish in China. Known for its rapid growth rate, strong disease resistance, and high nutritional value, it is considered a highly promising species for aquaculture. During the seed production process, it has been preliminarily observed that wild-caught broodstock exhibit superior reproductive performance compared to the first-generation (F1) artificially cultured broodstock. However, replacing wild-caught broodstock with artificially cultured broodstock for seed production is an inevitable trend and a necessary path for industry upgrading. By conducting in-depth research on the reproductive performance of wild and artificially cultured F1 generation Pharaoh cuttlefish broodstock, this study explores the aspects (spawning quantity and quality) and extent of differences in reproductive performance between them, providing a scientific theoretical basis for the development of the Pharaoh cuttlefish industry and promoting its growth. To clarify the differences in reproductive performance and offspring quality between wild and artificially cultured F1 generation Pharaoh cuttlefish broodstock, a controlled indoor experiment was conducted to compare their spawning quantity and quality, as well as the quality of newly hatched juvenile cuttlefish. Biochemical analyses were also performed to determine the composition and content of amino acids and fatty acids in the fertilized eggs produced by both groups. The results showed that the average number of eggs laid by wild broodstock was 2,050, while that of F1 artificially cultured broodstock was 1,306, indicating that wild broodstock produced 56.97% more eggs than F1 artificially cultured broodstock. The average fertilization rate of eggs produced by wild cuttlefish was 90.23%, whereas that of eggs produced by F1 artificially cultured broodstock was 54.05%, representing a 40.09% decrease compared to wild broodstock. The average long and short diameters of fertilized eggs produced by wild broodstock were 29.55 mm and 16.95 mm, respectively, while those produced by F1 artificially cultured broodstock were 23.15 mm and 16.17 mm, respectively, representing a 27.65% decrease compared to wild broodstock. The average malformation rate of newly hatched juvenile cuttlefish from fertilized eggs produced by wild broodstock was 9.86%, while that of F1 artificially cultured broodstock was 20.40%, representing a 51.76% increase compared to wild broodstock. The average survival rate of juvenile cuttlefish 30 days after hatching from fertilized eggs produced by wild broodstock was 90.54%, while that of F1 artificially cultured broodstock was 81.25%, representing a 10.26% decrease compared to wild broodstock. Biochemical analyses of amino acids and fatty acids revealed that the content of several essential amino acids (such as Lyc, Met, The, Phe) and polyunsaturated fatty acids (such as C20:4n-6, C20:5n-3, and C22:6n-3) in fertilized eggs produced by F1 artificially cultured broodstock was significantly lower than in those produced by wild broodstock ( p < 0.05 ), indicating that these nutrients play an important role in the quantity and quality of eggs produced by F1 generation Pharaoh cuttlefish broodstock and the quality of hatched juvenile cuttlefish. In summary, wild Pharaoh cuttlefish broodstock outperform artificially cultured F1 generation broodstock in terms of spawning quantity, quality, and offspring quality. The differences in amino acids and fatty acids in the fertilized eggs produced by the two groups suggest that the quantity and quality of eggs produced by artificially cultured Pharaoh cuttlefish can be improved by optimizing the nutrition of F1 generation broodstock (e.g., by feeding diets rich in Lyc, Met, The, Phe, C18:2n-6, C20:4n-6, C20:5n-3, and C22:6n-3). cuttlefish Pharaoh cuttlefish wild-caught broodstocks reproductive performance hatching rate 1. Introduction Aquaculture is one of the fastest-growing global production sectors and has accounted for a major share of total global fisheries production in recent years, highlighting its vital role in food security and economic stability. While the domestication and genetic improvement of most aquatic animals remain at a relatively early stage compared to terrestrial animals, short-term cultured aquatic species possess several important characteristics, such as rich genetic diversity, external fertilization, and high reproductive performance. These traits allow for high gains in economic characteristics through selective breeding (Houston et al., 2020). Improvements in artificial reproduction technologies, along with the ease of transporting fertilized eggs and fry, have enabled the rapid and widespread dissemination of improved breeds, thereby exerting immediate and substantial positive impacts on production (Gratacap et al., 2019). The Pharaoh cuttlefish, commonly known as the pharaoh cuttlefish, belongs to the phylum Mollusca, class Cephalopoda, order Sepioidea, family Sepiidae, and genus Sepia. It is a warm-water, marine benthic cephalopod inhabiting tropical and subtropical regions, found at depths of 15 to 100 meters. Its distribution includes the Philippine Islands, the Malay Archipelago, northern, eastern, and western Australia, offshore areas of India, the Red Sea, and offshore regions of the South China Sea (Hi et al., 2005; Norman et al., 2000). In recent years, the natural resources of Pharaoh cuttlefish have been declining due to continuous environmental degradation and overfishing. Recognized for its fast growth rate (reaching 500 g in three months under cultivation), strong disease resistance, and high nutritional value, it is considered a highly promising species for aquaculture (Minton et al., 2001). After years of research, breakthroughs have been made in seedling cultivation and farming techniques, with successful breeding achieved in marine net cages and artificial environments.However, in the current breeding process of Pharaoh cuttlefish, wild-caught populations are primarily used as broodstock, which to some extent hinders the sustainable development of its aquaculture. Studies have identified certain differences in reproductive performance between artificially cultured and wild-caught broodstock, with wild populations generally yielding higher numbers than cultured ones (Palma et al., 2023; Matson et al., 2005). Moreover, the use of wild broodstock increases the risk of introducing pathogens into farming systems (Falahatkar et al., 2025). Additionally, the increasing capture of mature individuals from wild populations for breeding purposes may impact the replenishment of natural resources, ultimately affecting population dynamics and fisheries production.Utilizing artificially domesticated populations as broodstock for seedling production is key to overcoming these limitations. This approach also facilitates the selection of desirable genetic traits and the development of superior varieties with high resistance and resilience to environmental stressors (Niu et al., 2024; Xiaoying et al., 2023; Liu et al., 2024). The production and utilization of domesticated broodstock represent a crucial first step in establishing a breeding program. Therefore, in the development of the Sepia pharaonis aquaculture industry, replacing wild-caught populations with cultured ones as broodstock is an inevitable trend and a necessary path to promote industrial growth. By comparing the reproductive performance, quality, quantity, and biochemical composition of offspring from cultured and wild populations, we can quantify the cumulative genetic progress of important economic traits in genetic improvement programs and comprehensively evaluate their aquaculture performance. However, there is still a lack of research data on the reproductive biology of both wild and artificially bred populations of Sepia pharaonis. Thus, this study aims to evaluate the reproductive performance and offspring quality of cultured Pharaoh cuttlefish and compare them with those of wild-caught cuttlefish broodstock. The findings are expected to provide fundamental data and scientific basis for the future large-scale breeding and sustainable development of Sepia pharaonis. 2. Materials and Methods 2.1 Experimental Materials 2.1.1 Ource and culture of broodstock The wild Sepia pharaonis broodstock used in this experiment were captured from natural seawaters. The body weight of males was 2.78 ± 0.34 kg, and that of females was 1.66 ± 0.12 kg. The body weight of the artificially cultured F1 generation broodstock was 2.65 ± 0.34 kg for males and 1.59 ± 0.09 kg for females. During the experimental culture period, the water parameters were maintained as follows: temperature 24.0 ± 1.0 °C, water depth 1.2 m, salinity 24.5 ± 0.5, dissolved oxygen 6.25 ± 0.15 mg/L, pH 8.02 ± 0.06, and ammonia nitrogen content 0.05 ± 0.01 mg/L. The broodstock were fed frozen whiteleg shrimp (Litopenaeus vannamei) twice daily (at 08:00 and 16:00) until satiation. Cleaning and water exchange were conducted once per day, with 80% of the water volume replaced. After six days of acclimation, mating and spawning began. Thirty healthy F1 generation broodstock (15 males and 15 females) with intact body shape and good vitality were selected. They were cultured at a male-to-female ratio of 1:1 and a density of 2 individuals/m². 2.1.2 Collection of Fertilized Eggs In the F1 generation broodstock culture tanks, egg attachment substrates were provided. The Pharaoh cuttlefish exhibits intermittent terminal spawning, characterized by batch-synchronous spawning with a single spawning bout. Eggs are laid in batches over a spawning period of 30–60 days. Fertilized eggs were collected every five days. Unfertilized eggs (identified by the absence of a yolk sac) were sorted out, and the fertilization rate was calculated. The body weight, long diameter, and short diameter of the fertilized eggs, as well as the long and short diameters of the yolk sac, were measured. This process continued until no fertilized eggs were observed in the culture tanks for five consecutive days. During the spawning period, some F1 generation broodstock died due to energy depletion; deceased individuals were promptly removed from the tanks. 2.2 Research methods 2.2.1 Comparison of fertilized egg quality A total of 90 fertilized eggs from both wild and artificially cultured F1 generation cuttlefish broodstock were randomly selected during the same time period and placed in hatching frames. Each frame contained 30 fertilized eggs, and all frames were introduced into the same incubation tank for hatching. The incubation conditions were maintained as follows: water temperature 24.0 ± 0.5 °C, salinity 24.5 ± 0.5, dissolved oxygen 6.46 ± 0.24 mg/L, pH 8.02 ± 0.05, and ammonia nitrogen content 0.02 ± 0.01 mg/L. A daily water exchange of 50% was performed. Eggs were sampled and observed every 6 hours to record embryonic development. Water temperature, salinity, pH, and dissolved oxygen were measured daily at 09:00 and 16:00. Dead fertilized eggs were promptly removed to prevent water quality deterioration. The incubation period, hatching rate, deformity rate, body weight of hatchlings, and mantle length were recorded. 2.2.2 Comparison of hatchling body weight After all cuttlefish larvae had hatched, 100 hatchlings from both wild and artificially cultured fertilized eggs were randomly selected. For convenient recording and observation, the cuttlefish were reared in frames for 30 days. During the rearing period, the following conditions were maintained: water temperature 22.0 ± 1.0 °C, water depth 1.2 m, salinity 28.5 ± 0.5, dissolved oxygen 6.25 ± 0.15 mg/L, pH 8.02 ± 0.06, and ammonia nitrogen content 0.05 ± 0.01 mg/L. For the first two days, they were fed an adequate amount of Artemia nauplii. From the third day onward, they were switched to a diet of mysid shrimp. After satiation, any excess mysids and debris were removed. When the cuttlefish reached a mantle length of 1 cm, their diet was changed to frozen small shrimp. Water exchange was performed every three days, with 50% of the volume replaced. Water temperature, salinity, pH, and dissolved oxygen were measured and recorded daily, and any deceased cuttlefish were promptly removed. 2.3 Chemical analysis 2.3.1 Amino acid analysis The amino acid composition of cuttlefish fertilized eggs was analyzed using the following procedure. Samples of fertilized eggs (approximately 0.1 g dry weight) were accurately weighed and transferred into 40 mL hydrolysis tubes. Each tube received 8 mL of 6.0 M hydrochloric acid (HCl) solution. The tubes were then evacuated, purged with nitrogen, and heated at 110°C for 24 hours to complete hydrolysis. After hydrolysis, the reaction mixtures were diluted to a final volume of 50 mL with distilled water, centrifuged at 5000 × g for 10 min, and filtered. A 1 mL aliquot of each hydrolysate was collected and dried under vacuum at 50°C to remove residual HCl. The dried residues were reconstituted in 2-5 mL of 0.02 M HCl, depending on the expected amino acid concentration. Finally, a 1 mL portion of the supernatant was analyzed using a Hitachi 835-50 amino acid analyzer (Hitachi, Ltd., Tokyo, Japan). Amino acids were identified and quantified by comparing their retention times and peak areas with those of authentic standards (Azrita et al., 2024). 2.3.2 Fatty acid profile Fatty acids were derivatized to methyl esters according to the BF₃-catalyzed method described in the Chinese National Standard GB/T 17376-2008 (Animal and vegetable fats and oils—Preparation of methyl esters of fatty acids). Briefly, the extracted lipids were redissolved in 4 mL of sodium hydroxide-methanol solution (0.5 mol/L) and refluxed at 80°C for 20 min. Then, 4 mL of boron trifluoride-methanol solution (14% w/v, ~1.4 kg·L⁻¹) was added, and the mixture was heated in a water bath for an additional 20 min. After cooling to room temperature, 4 mL of n-hexane was added, and the mixture was vortexed for 30 s. Saturated sodium chloride solution was added to bring the mixture to a defined volume. After phase separation, the organic layer was filtered through a 0.20 μm organic phase syringe filter and transferred into a thick-walled glass vial equipped with a polytetrafluoroethylene (PTFE)-lined cap. The n-hexane layer was then mixed with a 50 mg·L⁻¹ methyl nonadecanoate internal standard solution at a 1:1 (v/v) ratio in a GC vial for analysis.Fatty acids were identified by comparing their gas chromatography retention times with those of a certified 37-component fatty acid methyl ester (FAME) mix. Quantification was performed using methyl nonadecanoate (C19:0) as the internal standard. The analysis was carried out using an Agilent 7890B gas chromatography system coupled with a mass spectrometer (GC-MS) (Puebla et al., 2025). 2.4 Measured parameters Following the hatching of all cuttlefish larvae, the hatching time, hatching rate, fertilization rate, and deformity rate were recorded. The experiment was concluded one week after the last egg hatched, with no further hatching observed. Hatching time: The duration required for all normally fertilized eggs in the experimental group to complete hatching (i.e., larval emergence from the egg membrane). Hatching rate: (Number of hatched larvae / Total number of fertilized eggs) × 100%. Deformity rate: (Number of deformed larvae / Total number of hatched larvae) × 100%. Fertilization rate: (Number of fertilized eggs / Total number of eggs spawned) × 100%. 2.5 Statistical analysis All data are presented as mean ± standard deviation. Statistical analyses were performed using SPSS software (version 20.0). Differences among groups were assessed by one-way analysis of variance (ANOVA), followed by Duncan's multiple range test for post-hoc comparisons if a significant difference was detected. A p-value of less than 0.05 was considered statistically significant. 3. Results 3.1Reproductive performance 3.1.1Fecundity and fertilization rate As shown in Table 1, the fecundity and fertilization rate of wild cuttlefish broodstock were 2050 ± 223 eggs and 90.23 ± 2.65%, respectively. In contrast, the artificially cultured F1 generation broodstock exhibited significantly lower values, with 1306 ± 351 eggs and a fertilization rate of 54.05 ± 6.16% (p < 0.05). Compared to the wild broodstock, the F1 generation showed reductions of 35.50% in fecundity and 40.09% in fertilization rate. Tab. 1 Number of eggs laid and fertilization rate of wild and pond-reared cultured F1 generation broodstocks of cuttlefish Sepia pharaonis Evaluation Wild-caught Pond-reared Fertilization rate(%) 90.23±2.65 a 54.05±6.16 b Number of spawns(eggs) 2050±223 a 1306±351 b 3.1.2 Fertilized egg quality As shown in Table 2, the long and short diameters of fertilized eggs from wild Sepia pharaonis broodstock were 29.55 ± 0.65 mm and 16.95 ± 0.21 mm, respectively. In contrast, those from the artificially cultured F1 generation were 23.15 ± 0.77 mm and 16.17 ± 1.12 mm. Compared with the wild broodstock, the F1 generation showed a significant reduction of 21.66% in the long diameter (p < 0.05), while no significant difference was observed in the short diameter. The long and short diameters of the yolk sac in fertilized eggs from wild broodstock were 9.94 ± 0.38 mm and 7.87 ± 0.21 mm, respectively, whereas those from the F1 generation were 7.40 ± 0.10 mm and 5.12 ± 0.09 mm. Significant reductions of 25.55% and 34.94% were observed in the yolk sac long and short diameters of the F1 generation compared to the wild broodstock. The hatching rate of fertilized eggs was 88.20 ± 4.65% for the wild broodstock and 55.12 ± 1.77% for the F1 generation, indicating a 37.50% decrease in the F1 generation. After hatching, the deformity rate of newly hatched larvae from wild broodstock was 9.86 ± 0.38%, while that from the F1 generation was 20.40 ± 0.15%, representing a 51.76% increase in the deformity rate for the F1 generation. The incubation period was 16.9 ± 0.2 days for fertilized eggs from wild broodstock and 16.7 ± 1.1 days for those from the F1 generation, with no significant difference between the two groups (p> 0.05). Tab. 2 Quality of fertilized eggs laid by the wild-caught and pond-reared cultured F1 generation broodstocks of cuttlefish Sepia pharaonis Evaluation Wild-caught Pond-reared Long diameter of egg(mm) 29.55±0.65 a 23.15±0.77 b Short diameter of egg(mm) 16.95±0.21 16.74±1.12 Long diameter of yolk sac (mm) 9.94±0.38 a 7.40±0.10 b Short diameter of yolk sac(mm) 7.87±0.21 a 5.12±0.09 b Hatchability(%) 88.20±4.65 a 55.12±1.77 b Malformation rate(%) 9.86±0.38 a 20.40±0.15 b Incubation time(d) 16.9±0.2 16.7±1.1 3.1.3 Incubation period, larval size, and health status of fertilized eggs As shown in Table 3, the body weight and mantle length of newly hatched cuttlefish from wild broodstock were 0.20 ± 0.02 g and 0.95 ± 0.02 cm, respectively. In contrast, those from the artificially cultured F1 generation broodstock were 0.16 ± 0.02 g and 0.83 ± 0.02 cm. Compared with the wild broodstock, the F1 offspring exhibited significant reductions of 20.00% in body weight and 12.63% in mantle length.The 30-day survival rate of larvae from wild broodstock was 90.54 ± 3.27%, while that from the F1 generation was 81.25 ± 3.65%, representing a 10.26% decrease in the F1 generation. The weight gain rate was 65.67% for larvae derived from wild broodstock, compared to 49.96% for those from the F1 generation.No significant difference was observed in the incubation period of fertilized eggs between the wild and artificially cultured F1 generation broodstock (P> 0.05). Tab. 3 Quality of hatched juveniles from fertilized eggs laid by the wild-caught and pond-reared cultured F1 generation broodstocks cuttlefish Sepia pharaonis Evaluation Wild-caught Pond-reared Weight of freshly hatched cuttlefish(g) 0.20 a ±0.01 0.17 b ±0.01 The body length of freshly hatched cuttlefish(mm) 0.95±0.02 a 0.82±0.02 b The survival rates on the thirtueth day after hatching (%) 90.54±3.27 a 81.25±3.65 b Weight gain rate(%) 65.67±5.07 a 49.96±2.82 b 3.2 A comparison of amino acid and fatty acid compositions in fertilized eggs from wild and cultured F1 Sepia pharaonis As shown in Table 4, the fertilized eggs from artificially cultured F1 Sepia pharaonis exhibited higher levels of valine, isoleucine, and leucine compared to those from wild broodstock, with only valine being significantly elevated (p < 0.05). In contrast, the remaining essential amino acids—including lysine, methionine, threonine, and phenylalanine—were more abundant in fertilized eggs from wild broodstock. Among these, threonine and phenylalanine contents were significantly higher in the wild group (p < 0.05).Leucine, lysine, and valine were identified as the predominant essential amino acids (EAAs), collectively accounting for 49.90% and 53.22% of the total EAAs in fertilized eggs from wild and F1 broodstock, respectively. Regarding non-essential amino acids (NEAAs), aspartic acid and glutamic acid were the most abundant, constituting 44.48% and 47.47% of the total NEAAs in the wild and F1 groups, respectively. Tab. 4 Amino acid content of fertilized eggs produced by the wild-caught and pond-reared cultured F1 generation broodstocks of cuttlefish Sepia pharaonis Amino acid Wild-caught Pond-reared t-test(P-value) Lys* 5.09±0.57 4.47±0.13 0.145 Met* 2.81±0.38 2.41±0.19 0.179 Val* 3.71±0.04 a 3.92±0.09 b 0.025 Thr* 5.63±0.10 a 4.31±0.23 b ＜0.001 Ile* 5.19±0.50 5.23±0.05 0.910 Leu* 7.56±0.13 8.04±0.34 0.087 Phe* 2.79±0.02 a 2.49±0.08 b 0.003 Tyr 3.14±0.04 a 2.95±0.07 b 0.013 Cys 0.97±0.05 0.93±0.03 0.291 Ser 4.63±0.21 4.62±0.10 0.962 Gly 1.17±0.06 1.33±0.12 0.101 Glu 9.99±0.68 a 11.71±0.34 b 0.017 AsP 7.28±0.21 7.09±0.15 0.270 His 1.54±0.18 1.24±0.11 0.067 Ala 2.35±0.21 2.32±0.10 0.853 Arg 4.94±0.20 a 4.48±0.16 b 0.035 Pro 2.83±0.11 2.93±0.11 0.301 TAA1 71.61±1.93 70.47±0.38 0.415 EAA2 32.78±1.73 a 30.87±1.11 b 0.025 NEAA3 38.83±1.92 39.60±1.27 0.449 Values are expressed as mean ± SD. 3.2.2 Analysis of fatty acid composition As shown in Table 5, fatty acid analysis revealed that the predominant fatty acids were C16:0, C18:0, C16:1n-7, C18:1n-9, C18:1n-7, C20:4n-6 (arachidonic acid, ARA), C20:5n-3 (eicosapentaenoic acid, EPA), and C22:6n-3 (docosahexaenoic acid, DHA). Among these, the contents of C18:0, C18:1n-9, and C18:1n-7 in fertilized eggs from the artificially cultured F1 generation were significantly higher than those from wild broodstock (P 0.05).Conversely, the levels of C16:1n-7, C20:4n-6 (ARA), and C20:5n-3 (EPA) were elevated in fertilized eggs from wild broodstock compared to the F1 generation, but these differences did not reach statistical significance (P> 0.05). In contrast, the content of C22:6n-3 (DHA) was significantly higher in the wild group (P< 0.05).Overall, fertilized eggs from the artificially cultured F1 generation exhibited significantly higher total saturated fatty acids (∑SFA) and total monounsaturated fatty acids (∑MUFA) (P< 0.05). However, the total n-3 polyunsaturated fatty acids (∑n-3 PUFA) were significantly lower (P< 0.05), and the DHA/EPA ratio was also significantly reduced (P 0.05). Tab. 5 Fatty acid content of fertilized eggs produced from the wild-caught and pond-reared cultured F1 generation broodstocks of cuttlefish Sepia pharaonis Fatty acids Wild-caugh Pond-reared t-test(P-value) C14:0 2.92± 0.03 2.94 ±0.07 0.731 C15:0 0.45 ±0.06 0.56 ±0.06 0.078 C16:0 24.39 ±2.30 26.88 ±0.58 0.143 C17:0 1.65± 0.12 1.58 ±0.03 0.387 C18:0 12.88 ±0.31 a 14.76 ±0.82 b 0.021 C19:0 0.31± 0.02 0.32 ±0.03 0.738 C20:0 0.77 ±0.06 0.82 ±0.04 0.282 C22:0 0.25 ±0.04 0.28 ±0.04 0.435 C16: 1n-7 0.57 ±0.06 0.48 ±0.03 0.071 C18: 1n-9 4.22 ±0.05 a 4.62 ±0.10 b 0.003 C18:1n-7 0.90 ±0.02 a 1.00 ±0.05 b 0.035 C20:1n-9 1.68 ±0.07 1.56 ±0.11 0.179 C22:1n-9 0.28 ±0.02 0.29 ±0.03 0.887 C18:2n-6 0.43 ±0.03 0.46 ±0.05 0.492 C20:4n-6 8.38 ±0.43 7.45 ±0.42 0.054 C20:5n-3(EPA) 7.68 ±0.20 7.28 ±0.21 0.075 C22:6n-3(DHA) 32.27 ±1.67 a 28.83 ±0.84 b 0.033 ∑SFA 43.62 ±2.17 a 48.13 ±1.34 b 0.038 ∑MUFA 7.64 ±0.13 a 7.94 ±0.06 b 0.021 n-6 PUFA 8.82 ±0.45 7.91 ±0.39 0.057 n-3 PUFA 39.95 ±1.85 a 36.11 ±1.00 b 0.034 ∑PUFA 48.77 ±2.29 44.02 ±1.36 0.286 DHA/EPA 4.20 ±0.13 3.96 ±0.08 0.056 4. Discussion The Pharaoh cuttlefish is gonochoric, with most females dying after spawning due to energy depletion. Males can reach a maximum lifespan of up to 5 years, while both sexes reach sexual maturity within their first year of life. Reproductive performance serves as the foundation for population sustainability in aquatic animals, representing a critical indicator of productivity and a key trait reflecting their ability to adapt to environmental changes (Paulpand et al., 2024; Qiao et al., 2024). The reproductive performance of aquatic animals is influenced by numerous factors, including genetics, nutrition, physiology, and environmental conditions. Appropriate management practices and scientific feeding strategies can enhance reproductive outcomes, thereby generating greater economic returns (Engdaw et al., 2024; Atheesh et al., 2023). Key reproductive processes—such as gonadal development, fecundity, gamete production, fertilization rate, egg incubation, embryonic development, as well as larval survival and quality—are closely linked to dietary nutrient supply. Adequate nutrition promotes reproductive performance, whereas either nutrient excess or deficiency can impair reproductive capacity and even lead to mortality (Sika et al., 2023). Chronic exposure to poor rearing environments and nutritionally imbalanced diets can result in the decline of reproductive performance. These findings highlight the need for further research into optimal environmental conditions and dietary formulations for the culture of Sepia pharaonis.Studies on the whiteleg shrimp (Litopenaeus vannamei) have also demonstrated that certain female reproductive traits—such as egg number, hatching rate, and spawning frequency—are heritable, suggesting that spawning performance and offspring quality can be improved through genetic selection (Shi et al., 2024; Barral-Pintos et al., 2023). Furthermore, the rearing environment is a critical factor influencing reproductive performance. Adverse conditions such as elevated temperature, high humidity, and excessive noise can negatively impact the health of aquatic animals, thereby impairing their reproductive capacity (Bezerra et al., 2023; Satheesh et al., 2023). Studies indicate that deviations from natural temperature regimes are a major contributor to precocious puberty. Consistently high water temperatures, for instance, can stimulate gonadal development (Warren-Myers et al., 2020; Mlingi et al., 2025). Similarly, frequent water exchange may also accelerate gonadal maturation via neuroendocrine regulation. Once sexual maturity is prematurely reached, a phenomenon often described as "stunted growth" occurs, characterized by markedly slowed or ceased body weight gain. Consequently, environmental factors, particularly temperature, are primary reasons for the significantly smaller body size of sexually mature individuals in cultured populations compared to their wild counterparts (Nur et al., 2019; Callam et al., 2016; Stahlschmidt et al., 2015; Tokranov et al., 2024), ultimately affecting the reproductive performance of Sepia pharaonis.Precocious maturation under artificial cultivation has also been observed in other aquatic species, such as the Chinese sturgeon (Acipenser sinensis) (Jia et al., 2024), the Chinese mitten crab (Eriocheir sinensis) (Beckman et al., 2007), and the large yellow croaker (Larimichthys crocea) (Zhang et al., 2020), consistently resulting in a smaller body size at maturity in farmed stocks. As a benthic marine organism, the Pharaoh cuttlefish experiences a substantial disparity between current artificial farming facilities and its natural habitat, especially regarding light and temperature regimes.Age is another significant factor affecting reproductive performance in aquatic animals. Research has shown that reproductive performance in some fish species is relatively poor from birth until two years of age but begins to stabilize and improve after three years (Satheesh et al., 2023; Otoh et al., 2024). The present study revealed substantial differences in reproductive performance between cultured and wild populations of Sepia pharaonis. Specifically, the cultured populations exhibited significant reductions of 56.97% in fecundity and 40.09% in fertilization rate compared to their wild counterparts. Similar phenomena have been documented in the reproductive biology of the Chinese sturgeon (Acipenser sinensis), where cultured individuals show declining trends in both fecundity and reproductive success, posing considerable challenges for conservation breeding programs.Individual reproductive fitness is determined by the interplay of genetic predisposition and environmental conditions, closely associated with external factors such as habitat quality, geographical distribution, nutritional status, and physiological characteristics. Therefore, these factors may collectively contribute to the observed precocious maturation in cultured Sepia pharaonis, resulting in smaller body sizes of both broodstock and offspring in artificial breeding systems compared to wild populations.Consequently, future breeding programs should prioritize stringent control of environmental parameters—particularly salinity and temperature—to mitigate their adverse effects on reproductive performance. Such measures are essential for improving the overall quality and productivity of cultured Sepia pharaonis populations. Compared with wild cuttlefish eggs, the fertilized eggs of cultured Sepia pharaonis exhibited higher levels of valine, isoleucine, and leucine; however, only valine was significantly elevated (p < 0.05). In contrast, the remaining essential amino acids—including lysine, methionine, threonine, and phenylalanine—were more abundant in wild eggs. Among these, threonine and phenylalanine contents were significantly higher in the wild group (p < 0.05).Studies have indicated that amino acid profiles play a critical role in physiological condition and reproductive performance. Inadequate dietary protein supply in aquaculture has been shown to significantly reduce hatching rates and overall reproductive output. For instance, methionine (Met) is directly involved in spermatogenesis; low methionine levels can impair sperm quality and reduce the number of viable spermatozoa, leading to decreased fertilization and hatching rates, thereby adversely affecting cuttlefish reproductive performance (Life Science Research - Aquaculture, 2020).Therefore, supplementing broodstock diets with optimal levels of key amino acids may represent a viable strategy to enhance reproductive performance and offspring quality in future breeding programs. In the present study, fertilized eggs from the artificially cultured F1 generation of Sepia pharaonis exhibited significantly higher levels of C18:0, C18:1n-9, and C18:1n-7 compared to those from wild broodstock ( p 0.05). Conversely, significant decreases were observed in the F1 generation for the EPA/DHA ratio, C20:4n-6 (ARA) content, and total n-3 PUFA levels compared to the wild group ( p < 0.05 ).It has been reported that highly unsaturated fatty acids (HUFAs) play a crucial role in maintaining normal sexual maturation in aquatic broodstock, promoting vitellogenesis and embryonic development, and improving egg hatching rates (Callan et al., 2014; Bonvini et al., 2015). In several aquatic species, eggs with higher HUFA content often demonstrate higher hatching rates and/or shorter embryonic development periods, as observed in the whiteleg shrimp, Litopenaeus vannamei (Cardona et al., 2020), the yellow catfish, Pelteobagrus fulvidraco (Cahu et al., 2020), and the striped catfish, Pangasianodon hypophthalmus (Shuzhan et al., 2016).The synthesis of yolk and embryonic development in aquatic animals require adequate HUFAs. During gonadal maturation, HUFAs serve not only as an energy source (Amran et al., 2021) but also as essential nutrients for continuous gonadal development and embryonic morphogenesis, along with other critical components such as essential fatty acids, phospholipids, and specific hormones. It has long been recognized that aquatic animals possess little or no capacity for the de novo synthesis of n-3 HUFAs (Wu et al., 2024; Duc et al., 2023; Hideaki et al., 2025). Therefore, future aquaculture practices should focus on optimizing broodstock nutrition by supplementing diets with appropriate levels of fatty acids and regulating the content and ratios of amino and fatty acids to enhance reproductive performance and offspring quality.In summary, key indicators for assessing fertilized egg quality include egg viability, hatching rate, and the size and health status of newly hatched larvae (Caley et al., 2024). In the present study, fertilized eggs from the cultured population exhibited significantly lower values in egg size, hatching rate, larval size, and larval health metrics compared to those from the wild population. The artificially bred Sepia pharaonis showed reduced reproductive performance, with wild eggs yielding higher-quality hatchlings—evidenced by superior spawning output, hatching rates, embryonic development efficiency, and larval survival—which are also influenced by the content of essential fatty acids and amino acids.Given the current challenges in conservation breeding programs, it is imperative to further optimize environmental conditions (e.g., temperature, light), refine dietary formulations, and strengthen husbandry management practices (e.g., water quality control, feeding regimes) to improve the reproductive performance of artificially propagated Sepia pharaonis populations. References Houston, R.D., Bean, T.P., Macqueen, D.J., Gundappa, M.K., Jin, Y.H., Jenkins, T.L.,Selly, S.L.C., Martin, S.A., Stevens, J.R., Santos, E.M., 2020. Harnessing genomics to fast-track genetic improvement in aquaculture. Nat. Rev. Genet. 21, 389–409. Gratacap, R.L., Wargelius, A., Edvardsen, R.B., Houston, R.D., 2019. Potential of genome editing to improve aquaculture breeding and production. Trends Genet. 35, 672–684. https://doi.org/10.1016/j.tig.2019.06.006. hi W ,Zha S ,Hu L , et al. Ageing reduces the reproductive performance of male white shrimp Litopenaeus vannamei by altering sperm intracellular Ca 2+ concentrations and interfering with sperm apoptosis [J]. Animal Reproduction Science, 2018, 198 74-81. Jereb P，Ｒoper C F E． Cephalopods of the world［M］．Ｒome: Food and Agriculture Organization of the United Nations，2005: 106 － 108． Norman M，Ｒeid A． Pharaoh's cuttlefish，Sepia pharaonis． a guide to squid，cuttlefish，and octopuses of Australasia［M］． CSIＲO Publishing: Collingwood，Victoria，2000，29． Minton J W，Walsh L S，Lee P G，et al． First multi-generation culture of the tropical cuttlefish Sepia pharaonis Ehrenberg，1831 ［J］． Aquaculture International，2001，9( 5) : 375 － 392． Palma J ,Andrade P J ,Bureau P D . Growth, Reproductive Performances, and Brood Quality of Long Snout Seahorse, Hippocampus guttulatus , Fed Enriched Shrimp Diets [J]. Journal of the World Aquaculture Society, 2012, 43 (6): 802-813. Shyne S P A ,P. C B ,R. A , et al. Reproductive performance of captive-reared Indian white shrimp, Penaeus indicus, broodstocks over two generations [J]. Frontiers in Marine Science, 2023, Matson E S ,Langdon J C ,Evans S . Specific pathogen free culture of the Pacific oyster ( Crassostrea gigas ) in a breeding research program: Effect of water treatment on growth and survival [J]. Aquaculture, 2005, 253 (1): 475-484. Falahatkar B ,Efatpanah I ,Kargar R E , et al. Domestication may affect spawning performance of F1 pikeperch (Sander lucioperca) during consecutive captive reproduction [J]. Aquaculture Reports, 2025, 40 102561-102561. Niu X ,Lin L ,Zhang T , et al. Comparison of the intestinal flora of wild and artificial breeding green turtles (Chelonia mydas) [J]. Frontiers in Microbiology, 2024, 15 1412015-1412015. Xiaoying C ,Minwei H ,Kai P , et al. Artificial spawning, hatchery and culture of the tropical sea cucumber (Holothuria leucospilota) using non-living food [J]. Aquaculture, 2023, 575 Liu H ,Li P ,Leng X , et al. Exploring the adaptive behaviour and environmental acclimation of artificially-bred Chinese sturgeon (Acipenser sinensis) in semi-open marine environment: insights for endangered species conservation [J]. Reviews in Fish Biology and Fisheries, 2024, (prepublish): 1-21. Azrita A ,Syandri H ,Zakeri H , et al. Analysis of Fatty Acids and Amino Acids of Three Local Freshwater Bagridae Fish Species in the Kampar Kanan River, Indonesia, for Food Security. [J]. International journal of food science, 2024, 2024 6639837-6639837. Puebla L S ,Santé A F M ,Romero J , et al. Analysis of Fatty Acid Profile, α-Tocopherol, Squalene and Cholesterol Content in Edible Parts and By-Products of South Pacific Wild Fishes [J]. Marine Drugs, 2025, 23 (3): 104-104. Jia X ,Jiang W ,Zhou R , et al. Molecular mechanisms underlying sexual precocity in Chinese mitten crab (Eriocheir sinensis): aldh1a1 regulates retinol metabolism via miRNAs in the hepatopancreas [J]. Aquaculture Reports, 2024, 39 102460-102460. Beckman R B ,Gadberry B ,Parkins P , et al. State-dependent life history plasticity in Sacramento River winter-run chinook salmon (Oncorhynchus tshawytscha): interactions among photoperiod and growth modulate smolting and early male maturation [J]. Canadian Journal of Fisheries and Aquatic Sciences, 2007, 64 (2): 256-271. Zhang Z ,Bai Q ,Xu X , et al. Effects of environmental enrichment on the welfare of juvenile black rockfish Sebastes schlegelii : Growth, behavior and physiology [J]. Aquaculture, 2020, 518 734782-734782. Nur A M M ,Muizelaar W ,Boelen P , et al. Environmental and nutrient conditions influence fucoxanthin productivity of the marine diatom Phaeodactylum tricornutum grown on palm oil mill effluent [J]. Journal of Applied Phycology, 2019, 31 (1): 111-122. Callam R B ,Allen K S ,Frank-Lawale A . Genetic and environmental influence on triploid Crassostrea virginica grown in Chesapeake Bay: Growth [J]. Aquaculture, 2016, 452 97-106. Stahlschmidt R Z ,Jodrey D A ,Luoma L R . Consequences of complex environments: Temperature and energy intake interact to influence growth and metabolic rate [J]. Comparative Biochemistry and Physiology, Part A, 2015, 187 1-7. Tokranov M A ,Emelin O P ,Orlov M A . Distribution and biology of understudied slender eelblenny Lumpenus fabricii (Stichaeidae, Teleostei) in two Siberian Arctic seas, with comparative data on some other ground fishes [J]. Polar Biology, 2024, 48 (1): 17-17. Paulpandi S ,Sathya S ,Velmurugan K . Enhancing Aquaculture Productivity through Integrated Farming Systems: A Comparative Study of Fish-Pig and Fish-Duck Systems [J]. Asian Journal of Environment & Ecology, 2024, 23 (11): 53-62. Qiao H ,Jiang S ,Fu H . Special Issue: Molecular Advance on Reproduction and Fertility of Aquatic Animals [J]. International Journal of Molecular Sciences, 2024, 25 (21): 11610-11610. Engdaw F ,Geremew A . Broodstock nutrition in Nile tilapia and its implications on reproductive efficiency [J]. Frontiers in Aquaculture, 2024, 3 atheesh M ,Hari G P ,Parimal S , et al. Dietary protein requirement of female climbing perch, Anabas testudineus (Bloch, 1792) broodstock [J]. Animal Feed Science and Technology, 2023, 305 Sika PEAN ,N'dri JK ,Amani CSN . Influence of Diet and Dietary Crude Protein Content on the Reproductive Performance of the Snail Archachatina marginata (Swainson, 1821) [J]. Journal of Agriculture and Ecology Research International, 2023, 24 (6): 14-20. Bezerra MV ,Reis APG ,Melo DLC , et al. A long-term high temperature on young Nile tilapia females affects its urogenital papilla morphology and future reproductive performance [J]. Aquaculture, 2025, 595 (P2): 741666-741666. Satheesh M ,Hari GP ,Parimal S , et al. Dietary protein requirement of female climbing perch, Anabas testudineus (Bloch, 1792) broodstock [J]. Animal Feed Science and Technology, 2023, 305 Caley A ,Marzinelli M E ,Byrne M , et al. Artificial light at night and warming impact grazing rates and gonad index of the sea urchin Centrostephanus rodgersii. [J]. Proceedings. Biological sciences, 2024, 291 (2021): 20240415-20240415. Warren-Myers F ,Swearer E S ,Overton K , et al. Stocking density and rearing environment affect external condition, gonad quantity and gonad grade in onshore sea urchin roe enhancement aquaculture [J]. Aquaculture, 2020, 515 734591-734591. Mlingi T F ,Puvanendran V ,Burgerhout E , et al. “Influence of short-to-continuous and continuous photoperiods combined with elevated temperatures on sexual maturation in lumpfish (Cyclopterus lumpus, L. 1758)” [J]. Aquaculture, 2025, 598 741981-741981. Shi M ,Jiang S ,Shi J , et al. Evaluation of Genetic Parameters and Comparison of Stress Tolerance Traits in Different Strains of Litopenaeus vannamei [J]. Animals, 2024, 14 (4): BarralPintos X ,Arévalo M ,Escalante K , et al. Sperm quality of Litopenaeus vannamei fed fresh or experimental food in two culture systems [J]. Journal of the World Aquaculture Society, 2023, 55 (2): Shuzhan F ,Zheng C ,Yu X , et al. Effects of dietary arachidonic acid on reproduction performance, tissue fatty acid profile and gonadal steroidogenesis in female yellow catfish Pelteobagrus fulvidraco [J]. Aquaculture Nutrition, 2020, 27 (3): 700-711. Pamungkas W ,Jusadi D ,Jr Z M , et al. Effect of dietary essential fatty acids on level of oestradiol‐17β and vitellogenin, reproductive performance and larval quality of striped catfish (Pangasianodon hypophthalmus) in out‐of‐spawning season [J]. Aquaculture Research, 2020, 51 (9): 3900-3909. Callan K C ,Laidley W C ,Kling J L , et al. The effects of dietary HUFA level on flame angelfish ( C entropyge loriculus ) spawning, egg quality and early larval characteristics [J]. Aquaculture Research, 2014, 45 (7): 1176-1186. Bonvini E ,Parma L ,Mandrioli L , et al. Feeding common sole ( Solea solea ) juveniles with increasing dietary lipid levels affects growth, feed utilization and gut health [J]. Aquaculture, 2015, 449 87-93. Cardona E ,Lorgeoux B ,Chim L , et al. Biofloc contribution to antioxidant defence status, lipid nutrition and reproductive performance of broodstock of the shrimp Litopenaeus stylirostris: Consequences for the quality of eggs and larvae [J]. Aquaculture, 2016, 452 252-262. Amran M A ,Ariffin H ,Rahim A A , et al. The effect of lipid level on the growth and reproductive performance of female orange mud crab, Scylla olivacea (Herbst, 1796), during the fattening period [J]. Aquaculture Nutrition, 2021, 27 (6): 2497-2513. Wu J ,Liu Y ,Wang Y , et al. The Influence of Dietary n-3 Highly Unsaturated Fatty Acids on Growth, Fatty Acid Profile, Lipid Metabolism, Inflammatory Response, and Intestinal Microflora in F2 Generation Female Yangtze Sturgeon (Acipenser dabryanus) [J]. Animals, 2024, 14 (23): 3523-3523. Duc H P ,A.B. M S ,Arifur M R , et al. Effects of n-3 HUFA-enriched Artemia on growth, biochemical response, skeletal morphology and stress resistance of Asian sea bass (Lates calcarifer) larvae reared at high temperature [J]. Aquaculture, 2023, 574 Hideaki M ,Shinji S ,Mayumi W , et al. Application of genetic disruption of a Nannochloropsis oceanica cell wall synthesizing gene to n-3 HUFA enrichment of Brachionus plicatilis [J]. Aquaculture, 2022, 552 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 08 Dec, 2025 Editor assigned by journal 08 Dec, 2025 Submission checks completed at journal 07 Dec, 2025 First submitted to journal 06 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8293058","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":557199901,"identity":"f1e931c3-eb2d-414b-a713-e448f03a3ca6","order_by":0,"name":"Jianbo Zhang","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Jianbo","middleName":"","lastName":"Zhang","suffix":""},{"id":557199904,"identity":"41fd166e-b65c-4706-bf03-c9c3a9e93ff7","order_by":1,"name":"Qilong Huang","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Qilong","middleName":"","lastName":"Huang","suffix":""},{"id":557199907,"identity":"146e006b-ee58-40d6-9c3c-35eb31ac7749","order_by":2,"name":"Jiayi Sun","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Jiayi","middleName":"","lastName":"Sun","suffix":""},{"id":557199911,"identity":"67389d0e-a4c3-486c-a1ec-0557e3fdfdb2","order_by":3,"name":"Jiawen Yu","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Jiawen","middleName":"","lastName":"Yu","suffix":""},{"id":557199912,"identity":"35a54bbb-edba-4da6-a758-12002ddfe31d","order_by":4,"name":"Ruibing Peng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYPACiQQ2BgbGB4wNIE4C8VqYDUjRAlbGJkGUFnP23sOvK9ss8vik269VF+44zMDPnmPA8HMHbi2WPefSLM+2SRSzyZwpuz3zzGEGyZ43Boy9Z3BrMbiRY2bY2CaR2CaRk3abt+0wSMSAmbENj5b7bxBaikFa7AlqucFj/BCiJf0YM9gWCQJaLHtyzBgbzgH9IpHDLM17Jp1H4syzgoO9eLSYs58x/thQVpcnPyP94WfeHdZy/O3JGx/8xOcwUHRAmDwGYBJEHMCtAayF+QOEyf4An8JRMApGwSgYwQAAVphOutGRY4IAAAAASUVORK5CYII=","orcid":"","institution":"Ningbo University","correspondingAuthor":true,"prefix":"","firstName":"Ruibing","middleName":"","lastName":"Peng","suffix":""},{"id":557199913,"identity":"e675c020-bd32-40b0-8eb5-a421675eb58d","order_by":5,"name":"Maowang Jiang","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Maowang","middleName":"","lastName":"Jiang","suffix":""},{"id":557199916,"identity":"ebc2651d-b5f5-490d-b8e7-1b79782704a9","order_by":6,"name":"Qingxi Han","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Qingxi","middleName":"","lastName":"Han","suffix":""},{"id":557199917,"identity":"cdfe6144-59e2-41c4-88f1-1b998043cbe1","order_by":7,"name":"Xiamin Jiang","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Xiamin","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2025-12-06 08:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8293058/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8293058/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100155961,"identity":"f7b9cbaa-1a3a-4d63-8dce-4cd7b6e04cd1","added_by":"auto","created_at":"2026-01-13 14:10:24","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":40086,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8293058/v1/1750f71c0d6a2ffb34fbf18d.docx"},{"id":100155970,"identity":"7c929c8e-f56a-4278-ba8d-aea2902e8131","added_by":"auto","created_at":"2026-01-13 14:10:26","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11046,"visible":true,"origin":"","legend":"","description":"","filename":"3a3a4e3134d048b99a7590146b988c7f.json","url":"https://assets-eu.researchsquare.com/files/rs-8293058/v1/8bbca120428ef626e242e056.json"},{"id":100155969,"identity":"097d7444-8152-4057-96f5-f850d822f957","added_by":"auto","created_at":"2026-01-13 14:10:25","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":119041,"visible":true,"origin":"","legend":"","description":"","filename":"3a3a4e3134d048b99a7590146b988c7f1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8293058/v1/2618bbe0ab3c06ab44804e5a.xml"},{"id":100155963,"identity":"f0da203d-05bc-43fc-b4da-eaf89f568e99","added_by":"auto","created_at":"2026-01-13 14:10:24","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116142,"visible":true,"origin":"","legend":"","description":"","filename":"3a3a4e3134d048b99a7590146b988c7f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8293058/v1/07ff17be94fe101e4b3e6a39.xml"},{"id":100368189,"identity":"e6941f43-f0cf-4153-bc2c-fab139b6ccdf","added_by":"auto","created_at":"2026-01-16 07:57:42","extension":"html","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127334,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8293058/v1/3ec7f509bb88f365ef2ef171.html"},{"id":100382354,"identity":"1c16a4e1-a282-4006-a24a-7d92c66970c1","added_by":"auto","created_at":"2026-01-16 10:42:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":784984,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8293058/v1/81986f96-f202-42e0-9b5c-2b15ad504cb6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative analysis of the reproductive performance and offspring quality of pond-reared and wild-caught pharaoh cuttlefish, Sepia pharaonis broodstock","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAquaculture is one of the fastest-growing global production sectors and has accounted for a major share of total global fisheries production in recent years, highlighting its vital role in food security and economic stability. While the domestication and genetic improvement of most aquatic animals remain at a relatively early stage compared to terrestrial animals, short-term cultured aquatic species possess several important characteristics, such as rich genetic diversity, external fertilization, and high reproductive performance. These traits allow for high gains in economic characteristics through selective breeding (Houston et al., 2020). Improvements in artificial reproduction technologies, along with the ease of transporting fertilized eggs and fry, have enabled the rapid and widespread dissemination of improved breeds, thereby exerting immediate and substantial positive impacts on production (Gratacap et al., 2019).\u003c/p\u003e\n\u003cp\u003eThe Pharaoh cuttlefish, commonly known as the pharaoh cuttlefish, belongs to the phylum Mollusca, class Cephalopoda, order Sepioidea, family Sepiidae, and genus Sepia. It is a warm-water, marine benthic cephalopod inhabiting tropical and subtropical regions, found at depths of 15 to 100 meters. Its distribution includes the Philippine Islands, the Malay Archipelago, northern, eastern, and western Australia, offshore areas of India, the Red Sea, and offshore regions of the South China Sea (Hi et al., 2005; Norman et al., 2000). In recent years, the natural resources of Pharaoh cuttlefish have been declining due to continuous environmental degradation and overfishing. Recognized for its fast growth rate (reaching 500 g in three months under cultivation), strong disease resistance, and high nutritional value, it is considered a highly promising species for aquaculture (Minton et al., 2001). After years of research, breakthroughs have been made in seedling cultivation and farming techniques, with successful breeding achieved in marine net cages and artificial environments.However, in the current breeding process of Pharaoh cuttlefish, wild-caught populations are primarily used as broodstock, which to some extent hinders the sustainable development of its aquaculture. Studies have identified certain differences in reproductive performance between artificially cultured and wild-caught broodstock, with wild populations generally yielding higher numbers than cultured ones (Palma et al., 2023; Matson et al., 2005). Moreover, the use of wild broodstock increases the risk of introducing pathogens into farming systems (Falahatkar et al., 2025). Additionally, the increasing capture of mature individuals from wild populations for breeding purposes may impact the replenishment of natural resources, ultimately affecting population dynamics and fisheries production.Utilizing artificially domesticated populations as broodstock for seedling production is key to overcoming these limitations. This approach also facilitates the selection of desirable genetic traits and the development of superior varieties with high resistance and resilience to environmental stressors (Niu et al., 2024; Xiaoying et al., 2023; Liu et al., 2024).\u003c/p\u003e\n\u003cp\u003eThe production and utilization of domesticated broodstock represent a crucial first step in establishing a breeding program. Therefore, in the development of the Sepia pharaonis aquaculture industry, replacing wild-caught populations with cultured ones as broodstock is an inevitable trend and a necessary path to promote industrial growth. By comparing the reproductive performance, quality, quantity, and biochemical composition of offspring from cultured and wild populations, we can quantify the cumulative genetic progress of important economic traits in genetic improvement programs and comprehensively evaluate their aquaculture performance. However, there is still a lack of research data on the reproductive biology of both wild and artificially bred populations of Sepia pharaonis. Thus, this study aims to evaluate the reproductive performance and offspring quality of cultured Pharaoh cuttlefish and compare them with those of wild-caught cuttlefish broodstock. The findings are expected to provide fundamental data and scientific basis for the future large-scale breeding and sustainable development of Sepia pharaonis.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Experimental Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.1 Ource and culture of broodstock\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wild Sepia pharaonis broodstock used in this experiment were captured from natural seawaters. The body weight of males was 2.78 \u0026plusmn; 0.34 kg, and that of females was 1.66 \u0026plusmn; 0.12 kg. The body weight of the artificially cultured F1 generation broodstock was 2.65 \u0026plusmn; 0.34 kg for males and 1.59 \u0026plusmn; 0.09 kg for females. During the experimental culture period, the water parameters were maintained as follows: temperature 24.0 \u0026plusmn; 1.0 \u0026deg;C, water depth 1.2 m, salinity 24.5 \u0026plusmn; 0.5, dissolved oxygen 6.25 \u0026plusmn; 0.15 mg/L, pH 8.02 \u0026plusmn; 0.06, and ammonia nitrogen content 0.05 \u0026plusmn; 0.01 mg/L. The broodstock were fed frozen whiteleg shrimp (Litopenaeus vannamei) twice daily (at 08:00 and 16:00) until satiation. Cleaning and water exchange were conducted once per day, with 80% of the water volume replaced. After six days of acclimation, mating and spawning began. Thirty healthy F1 generation broodstock (15 males and 15 females) with intact body shape and good vitality were selected. They were cultured at a male-to-female ratio of 1:1 and a density of 2 individuals/m\u0026sup2;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.2 Collection of Fertilized Eggs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the F1 generation broodstock culture tanks, egg attachment substrates were provided. The Pharaoh cuttlefish exhibits intermittent terminal spawning, characterized by batch-synchronous spawning with a single spawning bout. Eggs are laid in batches over a spawning period of 30\u0026ndash;60 days. Fertilized eggs were collected every five days. Unfertilized eggs (identified by the absence of a yolk sac) were sorted out, and the fertilization rate was calculated. The body weight, long diameter, and short diameter of the fertilized eggs, as well as the long and short diameters of the yolk sac, were measured. This process continued until no fertilized eggs were observed in the culture tanks for five consecutive days. During the spawning period, some F1 generation broodstock died due to energy depletion; deceased individuals were promptly removed from the tanks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Research methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1 Comparison of fertilized egg quality\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 90 fertilized eggs from both wild and artificially cultured F1 generation cuttlefish broodstock were randomly selected during the same time period and placed in hatching frames. Each frame contained 30 fertilized eggs, and all frames were introduced into the same incubation tank for hatching. The incubation conditions were maintained as follows: water temperature 24.0 \u0026plusmn; 0.5 \u0026deg;C, salinity 24.5 \u0026plusmn; 0.5, dissolved oxygen 6.46 \u0026plusmn; 0.24 mg/L, pH 8.02 \u0026plusmn; 0.05, and ammonia nitrogen content 0.02 \u0026plusmn; 0.01 mg/L. A daily water exchange of 50% was performed. Eggs were sampled and observed every 6 hours to record embryonic development. Water temperature, salinity, pH, and dissolved oxygen were measured daily at 09:00 and 16:00. Dead fertilized eggs were promptly removed to prevent water quality deterioration. The incubation period, hatching rate, deformity rate, body weight of hatchlings, and mantle length were recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2 Comparison of hatchling body weight\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter all cuttlefish larvae had hatched, 100 hatchlings from both wild and artificially cultured fertilized eggs were randomly selected. For convenient recording and observation, the cuttlefish were reared in frames for 30 days. During the rearing period, the following conditions were maintained: water temperature 22.0 \u0026plusmn; 1.0 \u0026deg;C, water depth 1.2 m, salinity 28.5 \u0026plusmn; 0.5, dissolved oxygen 6.25 \u0026plusmn; 0.15 mg/L, pH 8.02 \u0026plusmn; 0.06, and ammonia nitrogen content 0.05 \u0026plusmn; 0.01 mg/L. For the first two days, they were fed an adequate amount of Artemia nauplii. From the third day onward, they were switched to a diet of mysid shrimp. After satiation, any excess mysids and debris were removed. When the cuttlefish reached a mantle length of 1 cm, their diet was changed to frozen small shrimp. Water exchange was performed every three days, with 50% of the volume replaced. Water temperature, salinity, pH, and dissolved oxygen were measured and recorded daily, and any deceased cuttlefish were promptly removed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Chemical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1 Amino acid analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amino acid composition of cuttlefish fertilized eggs was analyzed using the following procedure. Samples of fertilized eggs (approximately 0.1 g dry weight) were accurately weighed and transferred into 40 mL hydrolysis tubes. Each tube received 8 mL of 6.0 M hydrochloric acid (HCl) solution. The tubes were then evacuated, purged with nitrogen, and heated at 110\u0026deg;C for 24 hours to complete hydrolysis. After hydrolysis, the reaction mixtures were diluted to a final volume of 50 mL with distilled water, centrifuged at 5000 \u0026times; g for 10 min, and filtered. A 1 mL aliquot of each hydrolysate was collected and dried under vacuum at 50\u0026deg;C to remove residual HCl. The dried residues were reconstituted in 2-5 mL of 0.02 M HCl, depending on the expected amino acid concentration. Finally, a 1 mL portion of the supernatant was analyzed using a Hitachi 835-50 amino acid analyzer (Hitachi, Ltd., Tokyo, Japan). Amino acids were identified and quantified by comparing their retention times and peak areas with those of authentic standards (Azrita et al., 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2 Fatty acid profile\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFatty acids were derivatized to methyl esters according to the BF₃-catalyzed method described in the Chinese National Standard GB/T 17376-2008 (Animal and vegetable fats and oils\u0026mdash;Preparation of methyl esters of fatty acids). Briefly, the extracted lipids were redissolved in 4 mL of sodium hydroxide-methanol solution (0.5 mol/L) and refluxed at 80\u0026deg;C for 20 min. Then, 4 mL of boron trifluoride-methanol solution (14% w/v, ~1.4 kg\u0026middot;L⁻\u0026sup1;) was added, and the mixture was heated in a water bath for an additional 20 min. After cooling to room temperature, 4 mL of n-hexane was added, and the mixture was vortexed for 30 s. Saturated sodium chloride solution was added to bring the mixture to a defined volume. After phase separation, the organic layer was filtered through a 0.20 \u0026mu;m organic phase syringe filter and transferred into a thick-walled glass vial equipped with a polytetrafluoroethylene (PTFE)-lined cap. The n-hexane layer was then mixed with a 50 mg\u0026middot;L⁻\u0026sup1; methyl nonadecanoate internal standard solution at a 1:1 (v/v) ratio in a GC vial for analysis.Fatty acids were identified by comparing their gas chromatography retention times with those of a certified 37-component fatty acid methyl ester (FAME) mix. Quantification was performed using methyl nonadecanoate (C19:0) as the internal standard. The analysis was carried out using an Agilent 7890B gas chromatography system coupled with a mass spectrometer (GC-MS) (Puebla et al., 2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Measured parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the hatching of all cuttlefish larvae, the hatching time, hatching rate, fertilization rate, and deformity rate were recorded. The experiment was concluded one week after the last egg hatched, with no further hatching observed.\u003c/p\u003e\n\u003cp\u003eHatching time:\u0026nbsp;The duration required for all normally fertilized eggs in the experimental group to complete hatching (i.e., larval emergence from the egg membrane).\u003c/p\u003e\n\u003cp\u003eHatching rate:\u0026nbsp;(Number of hatched larvae / Total number of fertilized eggs) \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003eDeformity rate:\u0026nbsp;(Number of deformed larvae / Total number of hatched larvae) \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003eFertilization rate:\u0026nbsp;(Number of fertilized eggs / Total number of eggs spawned) \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are presented as mean \u0026plusmn; standard deviation. Statistical analyses were performed using SPSS software (version 20.0). Differences among groups were assessed by one-way analysis of variance (ANOVA), followed by Duncan\u0026apos;s multiple range test for post-hoc comparisons if a significant difference was detected. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1Reproductive performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.1Fecundity and fertilization rate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 1, the fecundity and fertilization rate of wild cuttlefish broodstock were 2050 \u0026plusmn; 223 eggs and 90.23 \u0026plusmn; 2.65%, respectively. In contrast, the artificially cultured F1 generation broodstock exhibited significantly lower values, with 1306 \u0026plusmn; 351 eggs and a fertilization rate of 54.05 \u0026plusmn; 6.16% (p \u0026lt; 0.05). Compared to the wild broodstock, the F1 generation showed reductions of 35.50% in fecundity and 40.09% in fertilization rate.\u003c/p\u003e\n\u003cp\u003eTab. 1 Number of eggs laid and fertilization rate of wild and pond-reared cultured F1 generation broodstocks of cuttlefish \u003cem\u003eSepia pharaonis\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32px;\"\u003e\n \u003cp\u003eEvaluation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003eWild-caught\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003ePond-reared\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32px;\"\u003e\n \u003cp\u003eFertilization rate(%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e90.23\u0026plusmn;2.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e54.05\u0026plusmn;6.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 32px;\"\u003e\n \u003cp\u003eNumber of spawns(eggs)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e2050\u0026plusmn;223\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e1306\u0026plusmn;351\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.2 Fertilized egg quality\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 2, the long and short diameters of fertilized eggs from wild\u0026nbsp;Sepia pharaonis\u0026nbsp;broodstock were 29.55 \u0026plusmn; 0.65 mm and 16.95 \u0026plusmn; 0.21 mm, respectively. In contrast, those from the artificially cultured F1 generation were 23.15 \u0026plusmn; 0.77 mm and 16.17 \u0026plusmn; 1.12 mm. Compared with the wild broodstock, the F1 generation showed a significant reduction of 21.66% in the long diameter (p \u0026lt; 0.05), while no significant difference was observed in the short diameter.\u003c/p\u003e\n\u003cp\u003eThe long and short diameters of the yolk sac in fertilized eggs from wild broodstock were 9.94 \u0026plusmn; 0.38 mm and 7.87 \u0026plusmn; 0.21 mm, respectively, whereas those from the F1 generation were 7.40 \u0026plusmn; 0.10 mm and 5.12 \u0026plusmn; 0.09 mm. Significant reductions of 25.55% and 34.94% were observed in the yolk sac long and short diameters of the F1 generation compared to the wild broodstock.\u003c/p\u003e\n\u003cp\u003eThe hatching rate of fertilized eggs was 88.20 \u0026plusmn; 4.65% for the wild broodstock and 55.12 \u0026plusmn; 1.77% for the F1 generation, indicating a 37.50% decrease in the F1 generation. After hatching, the deformity rate of newly hatched larvae from wild broodstock was 9.86 \u0026plusmn; 0.38%, while that from the F1 generation was 20.40 \u0026plusmn; 0.15%, representing a 51.76% increase in the deformity rate for the F1 generation.\u003c/p\u003e\n\u003cp\u003eThe incubation period was 16.9 \u0026plusmn; 0.2 days for fertilized eggs from wild broodstock and 16.7 \u0026plusmn; 1.1 days for those from the F1 generation, with no significant difference between the two groups (p\u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003eTab. 2 \u0026nbsp;Quality of fertilized eggs laid by the wild-caught and pond-reared cultured F1 generation broodstocks of cuttlefish \u003cem\u003eSepia pharaonis\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eEvaluation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003eWild-caught\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003ePond-reared\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eLong diameter of egg(mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003e29.55\u0026plusmn;0.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e23.15\u0026plusmn;0.77\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eShort diameter of egg(mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003e16.95\u0026plusmn;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e16.74\u0026plusmn;1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eLong diameter of yolk sac (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003e9.94\u0026plusmn;0.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e7.40\u0026plusmn;0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eShort diameter of yolk sac(mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003e7.87\u0026plusmn;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e5.12\u0026plusmn;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eHatchability(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003e88.20\u0026plusmn;4.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e55.12\u0026plusmn;1.77\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eMalformation rate(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003e9.86\u0026plusmn;0.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e20.40\u0026plusmn;0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eIncubation time(d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003e16.9\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e16.7\u0026plusmn;1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.3 Incubation period, larval size, and health status of fertilized eggs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 3, the body weight and mantle length of newly hatched cuttlefish from wild broodstock were 0.20 \u0026plusmn; 0.02 g and 0.95 \u0026plusmn; 0.02 cm, respectively. In contrast, those from the artificially cultured F1 generation broodstock were 0.16 \u0026plusmn; 0.02 g and 0.83 \u0026plusmn; 0.02 cm. Compared with the wild broodstock, the F1 offspring exhibited significant reductions of 20.00% in body weight and 12.63% in mantle length.The 30-day survival rate of larvae from wild broodstock was 90.54 \u0026plusmn; 3.27%, while that from the F1 generation was 81.25 \u0026plusmn; 3.65%, representing a 10.26% decrease in the F1 generation. The weight gain rate was 65.67% for larvae derived from wild broodstock, compared to 49.96% for those from the F1 generation.No significant difference was observed in the incubation period of fertilized eggs between the wild and artificially cultured F1 generation broodstock (P\u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003eTab. 3 \u0026nbsp;Quality of hatched juveniles from fertilized eggs laid by the wild-caught and pond-reared cultured F1 generation broodstocks cuttlefish \u003cem\u003eSepia pharaonis\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eEvaluation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003eWild-caught\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003ePond-reared\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eWeight of\u0026nbsp;freshly hatched\u0026nbsp;cuttlefish(g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.20\u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e0.17\u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eThe body length of\u0026nbsp;freshly hatched\u0026nbsp;cuttlefish(mm)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.95\u0026plusmn;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e0.82\u0026plusmn;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eThe survival rates on the thirtueth day after hatching (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e90.54\u0026plusmn;3.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e81.25\u0026plusmn;3.65\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39px;\"\u003e\n \u003cp\u003eWeight gain rate(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003e65.67\u0026plusmn;5.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 33px;\"\u003e\n \u003cp\u003e49.96\u0026plusmn;2.82\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 A comparison of amino acid and fatty acid compositions in fertilized eggs from wild and cultured F1 \u003cem\u003eSepia pharaonis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 4, the fertilized eggs from artificially cultured F1 \u003cem\u003eSepia pharaonis\u003c/em\u003e exhibited higher levels of valine, isoleucine, and leucine compared to those from wild broodstock, with only valine being significantly elevated (p \u0026lt; 0.05). In contrast, the remaining essential amino acids\u0026mdash;including lysine, methionine, threonine, and phenylalanine\u0026mdash;were more abundant in fertilized eggs from wild broodstock. Among these, threonine and phenylalanine contents were significantly higher in the wild group (p \u0026lt; 0.05).Leucine, lysine, and valine were identified as the predominant essential amino acids (EAAs), collectively accounting for 49.90% and 53.22% of the total EAAs in fertilized eggs from wild and F1 broodstock, respectively. Regarding non-essential amino acids (NEAAs), aspartic acid and glutamic acid were the most abundant, constituting 44.48% and 47.47% of the total NEAAs in the wild and F1 groups, respectively.\u003c/p\u003e\n\u003cp\u003eTab. 4 \u0026nbsp;Amino acid content of fertilized eggs produced by the wild-caught and pond-reared cultured F1 generation broodstocks of cuttlefish \u003cem\u003eSepia pharaonis\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eAmino acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eWild-caught\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003ePond-reared\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003et-test(P-value)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eLys*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e5.09\u0026plusmn;0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e4.47\u0026plusmn;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.145\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eMet*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e2.81\u0026plusmn;0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e2.41\u0026plusmn;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.179\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eVal*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e3.71\u0026plusmn;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e3.92\u0026plusmn;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eThr*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 24px;\"\u003e\n \u003cp\u003e5.63\u0026plusmn;0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e4.31\u0026plusmn;0.23\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e＜0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eIle*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e5.19\u0026plusmn;0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e5.23\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.910\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eLeu* \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e7.56\u0026plusmn;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e8.04\u0026plusmn;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.087\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003ePhe*\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e2.79\u0026plusmn;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e2.49\u0026plusmn;0.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eTyr\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e3.14\u0026plusmn;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e2.95\u0026plusmn;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eCys\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e0.97\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.93\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.291\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eSer\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e4.63\u0026plusmn;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e4.62\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.962\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eGly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e1.17\u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e1.33\u0026plusmn;0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.101\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eGlu\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e9.99\u0026plusmn;0.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e11.71\u0026plusmn;0.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.017\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eAsP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e7.28\u0026plusmn;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e7.09\u0026plusmn;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.270\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eHis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e1.54\u0026plusmn;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e1.24\u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.067\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eAla\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e2.35\u0026plusmn;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e2.32\u0026plusmn;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.853\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eArg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e4.94\u0026plusmn;0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e4.48\u0026plusmn;0.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.035\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003ePro\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e2.83\u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e2.93\u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.301\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eTAA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e71.61\u0026plusmn;1.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e70.47\u0026plusmn;0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.415\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eEAA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e32.78\u0026plusmn;1.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e30.87\u0026plusmn;1.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003eNEAA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e38.83\u0026plusmn;1.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e39.60\u0026plusmn;1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25px;\"\u003e\n \u003cp\u003e0.449\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eValues are expressed as mean \u0026plusmn; SD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.2 Analysis of fatty acid composition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 5, fatty acid analysis revealed that the predominant fatty acids were C16:0, C18:0, C16:1n-7, C18:1n-9, C18:1n-7, C20:4n-6 (arachidonic acid, ARA), C20:5n-3 (eicosapentaenoic acid, EPA), and C22:6n-3 (docosahexaenoic acid, DHA). Among these, the contents of C18:0, C18:1n-9, and C18:1n-7 in fertilized eggs from the artificially cultured F1 generation were significantly higher than those from wild broodstock (P\u0026lt; 0.05). Although C16:0 was also higher in the F1 generation, the difference was not significant (P\u0026gt; 0.05).Conversely, the levels of C16:1n-7, C20:4n-6 (ARA), and C20:5n-3 (EPA) were elevated in fertilized eggs from wild broodstock compared to the F1 generation, but these differences did not reach statistical significance (P\u0026gt; 0.05). In contrast, the content of C22:6n-3 (DHA) was significantly higher in the wild group (P\u0026lt; 0.05).Overall, fertilized eggs from the artificially cultured F1 generation exhibited significantly higher total saturated fatty acids (\u0026sum;SFA) and total monounsaturated fatty acids (\u0026sum;MUFA) (P\u0026lt; 0.05). However, the total n-3 polyunsaturated fatty acids (\u0026sum;n-3 PUFA) were significantly lower (P\u0026lt; 0.05), and the DHA/EPA ratio was also significantly reduced (P\u0026lt; 0.05). Although the total n-6 PUFA (\u0026sum;n-6) content was lower in the F1 generation, this decrease was not statistically significant (P\u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003eTab. 5 Fatty acid content of fertilized eggs produced from the wild-caught and pond-reared cultured F1 generation broodstocks of cuttlefish \u003cem\u003eSepia pharaonis\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 27px;\"\u003e\n \u003cp\u003eFatty acids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eWild-caugh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003ePond-reared\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 26px;\"\u003e\n \u003cp\u003et-test(P-value)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC14:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e2.92\u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e2.94 \u0026plusmn;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.731\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC15:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.45 \u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.56 \u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.078\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC16:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e24.39 \u0026plusmn;2.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e26.88 \u0026plusmn;0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.143\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC17:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e1.65\u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e1.58 \u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.387\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC18:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e12.88 \u0026plusmn;0.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e14.76 \u0026plusmn;0.82\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC19:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.31\u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.32 \u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.738\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC20:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.77 \u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.82 \u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.282\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC22:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.25 \u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.28 \u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.435\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC16: 1n-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.57 \u0026plusmn;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.48 \u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.071\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC18: 1n-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e4.22 \u0026plusmn;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e4.62 \u0026plusmn;0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC18:1n-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.90 \u0026plusmn;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e1.00 \u0026plusmn;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.035\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC20:1n-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e1.68 \u0026plusmn;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e1.56 \u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.179\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC22:1n-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.28 \u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.29 \u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.887\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC18:2n-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.43 \u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e0.46 \u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.492\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC20:4n-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e8.38 \u0026plusmn;0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e7.45 \u0026plusmn;0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.054\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC20:5n-3(EPA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e7.68 \u0026plusmn;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e7.28 \u0026plusmn;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.075\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eC22:6n-3(DHA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e32.27 \u0026plusmn;1.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e28.83 \u0026plusmn;0.84\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.033\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003e\u0026sum;SFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e43.62 \u0026plusmn;2.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e48.13 \u0026plusmn;1.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.038\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003e\u0026sum;MUFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e7.64 \u0026plusmn;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e7.94 \u0026plusmn;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003en-6 PUFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e8.82 \u0026plusmn;0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e7.91 \u0026plusmn;0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.057\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003en-3 PUFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e39.95 \u0026plusmn;1.85\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e36.11 \u0026plusmn;1.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.034\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003e\u0026sum;PUFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e48.77 \u0026plusmn;2.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e44.02 \u0026plusmn;1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.286\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 27px;\"\u003e\n \u003cp\u003eDHA/EPA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e4.20 \u0026plusmn;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23px;\"\u003e\n \u003cp\u003e3.96 \u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 26px;\"\u003e\n \u003cp\u003e0.056\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe Pharaoh cuttlefish is gonochoric, with most females dying after spawning due to energy depletion. Males can reach a maximum lifespan of up to 5 years, while both sexes reach sexual maturity within their first year of life. Reproductive performance serves as the foundation for population sustainability in aquatic animals, representing a critical indicator of productivity and a key trait reflecting their ability to adapt to environmental changes (Paulpand et al., 2024; Qiao et al., 2024).\u003c/p\u003e\n\u003cp\u003eThe reproductive performance of aquatic animals is influenced by numerous factors, including genetics, nutrition, physiology, and environmental conditions. Appropriate management practices and scientific feeding strategies can enhance reproductive outcomes, thereby generating greater economic returns (Engdaw et al., 2024; Atheesh et al., 2023). Key reproductive processes\u0026mdash;such as gonadal development, fecundity, gamete production, fertilization rate, egg incubation, embryonic development, as well as larval survival and quality\u0026mdash;are closely linked to dietary nutrient supply. Adequate nutrition promotes reproductive performance, whereas either nutrient excess or deficiency can impair reproductive capacity and even lead to mortality (Sika et al., 2023). Chronic exposure to poor rearing environments and nutritionally imbalanced diets can result in the decline of reproductive performance. These findings highlight the need for further research into optimal environmental conditions and dietary formulations for the culture of\u0026nbsp;Sepia pharaonis.Studies on the whiteleg shrimp (Litopenaeus vannamei) have also demonstrated that certain female reproductive traits\u0026mdash;such as egg number, hatching rate, and spawning frequency\u0026mdash;are heritable, suggesting that spawning performance and offspring quality can be improved through genetic selection (Shi et al., 2024; Barral-Pintos et al., 2023).\u003c/p\u003e\n\u003cp\u003eFurthermore, the rearing environment is a critical factor influencing reproductive performance. Adverse conditions such as elevated temperature, high humidity, and excessive noise can negatively impact the health of aquatic animals, thereby impairing their reproductive capacity (Bezerra et al., 2023; Satheesh et al., 2023). Studies indicate that deviations from natural temperature regimes are a major contributor to precocious puberty. Consistently high water temperatures, for instance, can stimulate gonadal development (Warren-Myers et al., 2020; Mlingi et al., 2025). Similarly, frequent water exchange may also accelerate gonadal maturation via neuroendocrine regulation. Once sexual maturity is prematurely reached, a phenomenon often described as \u0026quot;stunted growth\u0026quot; occurs, characterized by markedly slowed or ceased body weight gain. Consequently, environmental factors, particularly temperature, are primary reasons for the significantly smaller body size of sexually mature individuals in cultured populations compared to their wild counterparts (Nur et al., 2019; Callam et al., 2016; Stahlschmidt et al., 2015; Tokranov et al., 2024), ultimately affecting the reproductive performance of\u0026nbsp;Sepia pharaonis.Precocious maturation under artificial cultivation has also been observed in other aquatic species, such as the Chinese sturgeon (Acipenser sinensis) (Jia et al., 2024), the Chinese mitten crab (Eriocheir sinensis) (Beckman et al., 2007), and the large yellow croaker (Larimichthys crocea) (Zhang et al., 2020), consistently resulting in a smaller body size at maturity in farmed stocks. As a benthic marine organism, the Pharaoh cuttlefish experiences a substantial disparity between current artificial farming facilities and its natural habitat, especially regarding light and temperature regimes.Age is another significant factor affecting reproductive performance in aquatic animals. Research has shown that reproductive performance in some fish species is relatively poor from birth until two years of age but begins to stabilize and improve after three years (Satheesh et al., 2023; Otoh et al., 2024).\u003c/p\u003e\n\u003cp\u003eThe present study revealed substantial differences in reproductive performance between cultured and wild populations of\u0026nbsp;Sepia pharaonis. Specifically, the cultured populations exhibited significant reductions of 56.97% in fecundity and 40.09% in fertilization rate compared to their wild counterparts. Similar phenomena have been documented in the reproductive biology of the Chinese sturgeon (Acipenser sinensis), where cultured individuals show declining trends in both fecundity and reproductive success, posing considerable challenges for conservation breeding programs.Individual reproductive fitness is determined by the interplay of genetic predisposition and environmental conditions, closely associated with external factors such as habitat quality, geographical distribution, nutritional status, and physiological characteristics. Therefore, these factors may collectively contribute to the observed precocious maturation in cultured\u0026nbsp;Sepia pharaonis, resulting in smaller body sizes of both broodstock and offspring in artificial breeding systems compared to wild populations.Consequently, future breeding programs should prioritize stringent control of environmental parameters\u0026mdash;particularly salinity and temperature\u0026mdash;to mitigate their adverse effects on reproductive performance. Such measures are essential for improving the overall quality and productivity of cultured\u0026nbsp;Sepia pharaonis\u0026nbsp;populations.\u003c/p\u003e\n\u003cp\u003eCompared with wild cuttlefish eggs, the fertilized eggs of cultured\u0026nbsp;Sepia pharaonis\u0026nbsp;exhibited higher levels of valine, isoleucine, and leucine; however, only valine was significantly elevated (p \u0026lt; 0.05). In contrast, the remaining essential amino acids\u0026mdash;including lysine, methionine, threonine, and phenylalanine\u0026mdash;were more abundant in wild eggs. Among these, threonine and phenylalanine contents were significantly higher in the wild group (p \u0026lt; 0.05).Studies have indicated that amino acid profiles play a critical role in physiological condition and reproductive performance. Inadequate dietary protein supply in aquaculture has been shown to significantly reduce hatching rates and overall reproductive output. For instance, methionine (Met) is directly involved in spermatogenesis; low methionine levels can impair sperm quality and reduce the number of viable spermatozoa, leading to decreased fertilization and hatching rates, thereby adversely affecting cuttlefish reproductive performance (Life Science Research - Aquaculture, 2020).Therefore, supplementing broodstock diets with optimal levels of key amino acids may represent a viable strategy to enhance reproductive performance and offspring quality in future breeding programs.\u003c/p\u003e\n\u003cp\u003eIn the present study, fertilized eggs from the artificially cultured F1 generation of\u0026nbsp;Sepia pharaonis\u0026nbsp;exhibited significantly higher levels of C18:0, C18:1n-9, and C18:1n-7 compared to those from wild broodstock (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e). Although the content of C16:0 was also elevated in the F1 generation, the difference was not statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026gt; 0.05). Conversely, significant decreases were observed in the F1 generation for the EPA/DHA ratio, C20:4n-6 (ARA) content, and total n-3 PUFA levels compared to the wild group (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e).It has been reported that highly unsaturated fatty acids (HUFAs) play a crucial role in maintaining normal sexual maturation in aquatic broodstock, promoting vitellogenesis and embryonic development, and improving egg hatching rates (Callan et al., 2014; Bonvini et al., 2015). In several aquatic species, eggs with higher HUFA content often demonstrate higher hatching rates and/or shorter embryonic development periods, as observed in the whiteleg shrimp,\u0026nbsp;Litopenaeus vannamei\u0026nbsp;(Cardona et al., 2020), the yellow catfish,\u0026nbsp;Pelteobagrus fulvidraco\u0026nbsp;(Cahu et al., 2020), and the striped catfish,\u0026nbsp;Pangasianodon hypophthalmus\u0026nbsp;(Shuzhan et al., 2016).The synthesis of yolk and embryonic development in aquatic animals require adequate HUFAs. During gonadal maturation, HUFAs serve not only as an energy source (Amran et al., 2021) but also as essential nutrients for continuous gonadal development and embryonic morphogenesis, along with other critical components such as essential fatty acids, phospholipids, and specific hormones. It has long been recognized that aquatic animals possess little or no capacity for the de novo synthesis of n-3 HUFAs (Wu et al., 2024; Duc et al., 2023; Hideaki et al., 2025).\u003c/p\u003e\n\u003cp\u003eTherefore, future aquaculture practices should focus on optimizing broodstock nutrition by supplementing diets with appropriate levels of fatty acids and regulating the content and ratios of amino and fatty acids to enhance reproductive performance and offspring quality.In summary, key indicators for assessing fertilized egg quality include egg viability, hatching rate, and the size and health status of newly hatched larvae (Caley et al., 2024). In the present study, fertilized eggs from the cultured population exhibited significantly lower values in egg size, hatching rate, larval size, and larval health metrics compared to those from the wild population. The artificially bred Sepia pharaonis showed reduced reproductive performance, with wild eggs yielding higher-quality hatchlings\u0026mdash;evidenced by superior spawning output, hatching rates, embryonic development efficiency, and larval survival\u0026mdash;which are also influenced by the content of essential fatty acids and amino acids.Given the current challenges in conservation breeding programs, it is imperative to further optimize environmental conditions (e.g., temperature, light), refine dietary formulations, and strengthen husbandry management practices (e.g., water quality control, feeding regimes) to improve the reproductive performance of artificially propagated Sepia pharaonis populations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHouston, R.D., Bean, T.P., Macqueen, D.J., Gundappa, M.K., Jin, Y.H., Jenkins, T.L.,Selly, S.L.C., Martin, S.A., Stevens, J.R., Santos, E.M., 2020. Harnessing genomics to fast-track genetic improvement in aquaculture. Nat. Rev. Genet. 21, 389\u0026ndash;409.\u003c/li\u003e\n \u003cli\u003eGratacap, R.L., Wargelius, A., Edvardsen, R.B., Houston, R.D., 2019. Potential of genome editing to improve aquaculture breeding and production. Trends Genet. 35, 672\u0026ndash;684. https://doi.org/10.1016/j.tig.2019.06.006.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ehi W ,Zha S ,Hu L , et al. Ageing reduces the reproductive performance of male white shrimp Litopenaeus vannamei by altering sperm intracellular Ca 2+ concentrations and interfering with sperm apoptosis [J]. Animal Reproduction Science, 2018, 198 74-81. Jereb P，Ｒoper C F E．\u0026nbsp;Cephalopods of the world［M］．Ｒome: Food and Agriculture Organization of the United Nations，2005: 106\u0026nbsp;－\u0026nbsp;108．\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eNorman M，Ｒeid A．\u0026nbsp;Pharaoh\u0026apos;s cuttlefish，Sepia pharaonis．\u0026nbsp;a guide to squid，cuttlefish，and octopuses of Australasia［M］．\u0026nbsp;CSIＲO Publishing: Collingwood，Victoria，2000，29．\u003c/li\u003e\n \u003cli\u003eMinton J W，Walsh L S，Lee P G，et al．\u0026nbsp;First multi-generation culture of the tropical cuttlefish Sepia pharaonis Ehrenberg，1831\u0026nbsp;［J］．\u0026nbsp;Aquaculture International，2001，9( 5) : 375\u0026nbsp;－\u0026nbsp;392．\u003c/li\u003e\n \u003cli\u003ePalma J ,Andrade P J ,Bureau P D . Growth, Reproductive Performances, and Brood Quality of Long Snout Seahorse, Hippocampus guttulatus , Fed Enriched Shrimp Diets [J]. Journal of the World Aquaculture Society, 2012, 43 (6): 802-813. \u0026nbsp; \u0026nbsp; \u0026nbsp; Shyne S P A ,P. C B ,R. A , et al. Reproductive performance of captive-reared Indian white shrimp, Penaeus indicus, broodstocks over two generations [J]. Frontiers in Marine Science, 2023,\u003c/li\u003e\n \u003cli\u003eMatson E S ,Langdon J C ,Evans S . Specific pathogen free culture of the Pacific oyster ( Crassostrea gigas ) in a breeding research program: Effect of water treatment on growth and survival [J]. Aquaculture, 2005, 253 (1): 475-484.\u003c/li\u003e\n \u003cli\u003eFalahatkar B ,Efatpanah I ,Kargar R E , et al. Domestication may affect spawning performance of F1 pikeperch (Sander lucioperca) during consecutive captive reproduction [J]. Aquaculture Reports, 2025, 40 102561-102561.\u003c/li\u003e\n \u003cli\u003eNiu X ,Lin L ,Zhang T , et al. Comparison of the intestinal flora of wild and artificial breeding green turtles (Chelonia mydas) [J]. Frontiers in Microbiology, 2024, 15 1412015-1412015.\u003c/li\u003e\n \u003cli\u003eXiaoying C ,Minwei H ,Kai P , et al. Artificial spawning, hatchery and culture of the tropical sea cucumber (Holothuria leucospilota) using non-living food [J]. Aquaculture, 2023, 575\u003c/li\u003e\n \u003cli\u003eLiu H ,Li P ,Leng X , et al. Exploring the adaptive behaviour and environmental acclimation of artificially-bred Chinese sturgeon (Acipenser sinensis) in semi-open marine environment: insights for endangered species conservation [J]. Reviews in Fish Biology and Fisheries, 2024, (prepublish): 1-21.\u003c/li\u003e\n \u003cli\u003eAzrita A ,Syandri H ,Zakeri H , et al. Analysis of Fatty Acids and Amino Acids of Three Local Freshwater Bagridae Fish Species in the Kampar Kanan River, Indonesia, for Food Security. [J]. International journal of food science, 2024, 2024 6639837-6639837.\u003c/li\u003e\n \u003cli\u003ePuebla L S ,Sant\u0026eacute; A F M ,Romero J , et al. Analysis of Fatty Acid Profile, \u0026alpha;-Tocopherol, Squalene and Cholesterol Content in Edible Parts and By-Products of South Pacific Wild Fishes [J]. Marine Drugs, 2025, 23 (3): 104-104.\u003c/li\u003e\n \u003cli\u003eJia X ,Jiang W ,Zhou R , et al. Molecular mechanisms underlying sexual precocity in Chinese mitten crab (Eriocheir sinensis): aldh1a1 regulates retinol metabolism via miRNAs in the hepatopancreas [J]. Aquaculture Reports, 2024, 39 102460-102460.\u003c/li\u003e\n \u003cli\u003eBeckman R B ,Gadberry B ,Parkins P , et al. State-dependent life history plasticity in Sacramento River winter-run chinook salmon (Oncorhynchus tshawytscha): interactions among photoperiod and growth modulate smolting and early male maturation [J]. Canadian Journal of Fisheries and Aquatic Sciences, 2007, 64 (2): 256-271.\u003c/li\u003e\n \u003cli\u003eZhang Z ,Bai Q ,Xu X , et al. Effects of environmental enrichment on the welfare of juvenile black rockfish Sebastes schlegelii : Growth, behavior and physiology [J]. Aquaculture, 2020, 518 734782-734782.\u003c/li\u003e\n \u003cli\u003eNur A M M ,Muizelaar W ,Boelen P , et al. Environmental and nutrient conditions influence fucoxanthin productivity of the marine diatom Phaeodactylum tricornutum grown on palm oil mill effluent [J]. Journal of Applied Phycology, 2019, 31 (1): 111-122.\u003c/li\u003e\n \u003cli\u003eCallam R B ,Allen K S ,Frank-Lawale A . Genetic and environmental influence on triploid Crassostrea virginica grown in Chesapeake Bay: Growth [J]. Aquaculture, 2016, 452 97-106.\u003c/li\u003e\n \u003cli\u003eStahlschmidt R Z ,Jodrey D A ,Luoma L R . Consequences of complex environments: Temperature and energy intake interact to influence growth and metabolic rate [J]. Comparative Biochemistry and Physiology, Part A, 2015, 187 1-7.\u003c/li\u003e\n \u003cli\u003eTokranov M A ,Emelin O P ,Orlov M A . Distribution and biology of understudied slender eelblenny Lumpenus fabricii (Stichaeidae, Teleostei) in two Siberian Arctic seas, with comparative data on some other ground fishes [J]. Polar Biology, 2024, 48 (1): 17-17.\u003c/li\u003e\n \u003cli\u003ePaulpandi S ,Sathya S ,Velmurugan K . Enhancing Aquaculture Productivity through Integrated Farming Systems: A Comparative Study of Fish-Pig and Fish-Duck Systems [J]. Asian Journal of Environment \u0026amp; Ecology, 2024, 23 (11): 53-62.\u003c/li\u003e\n \u003cli\u003eQiao H ,Jiang S ,Fu H . Special Issue: Molecular Advance on Reproduction and Fertility of Aquatic Animals [J]. International Journal of Molecular Sciences, 2024, 25 (21): 11610-11610.\u003c/li\u003e\n \u003cli\u003eEngdaw F ,Geremew A . Broodstock nutrition in Nile tilapia and its implications on reproductive efficiency [J]. Frontiers in Aquaculture, 2024, 3\u003c/li\u003e\n \u003cli\u003eatheesh M ,Hari G P ,Parimal S , et al. Dietary protein requirement of female climbing perch, Anabas testudineus (Bloch, 1792) broodstock [J]. Animal Feed Science and Technology, 2023, 305\u003c/li\u003e\n \u003cli\u003eSika PEAN ,N\u0026apos;dri JK ,Amani CSN . Influence of Diet and Dietary Crude Protein Content on the Reproductive Performance of the Snail Archachatina marginata (Swainson, 1821) [J]. Journal of Agriculture and Ecology Research International, 2023, 24 (6): 14-20.\u003c/li\u003e\n \u003cli\u003eBezerra MV ,Reis APG ,Melo DLC , et al. A long-term high temperature on young Nile tilapia females affects its urogenital papilla morphology and future reproductive performance [J]. Aquaculture, 2025, 595 (P2): 741666-741666.\u003c/li\u003e\n \u003cli\u003eSatheesh M ,Hari GP ,Parimal S , et al. Dietary protein requirement of female climbing perch, Anabas testudineus (Bloch, 1792) broodstock [J]. Animal Feed Science and Technology, 2023, 305\u003c/li\u003e\n \u003cli\u003eCaley A ,Marzinelli M E ,Byrne M , et al. Artificial light at night and warming impact grazing rates and gonad index of the sea urchin Centrostephanus rodgersii. [J]. Proceedings. Biological sciences, 2024, 291 (2021): 20240415-20240415.\u003c/li\u003e\n \u003cli\u003eWarren-Myers F ,Swearer E S ,Overton K , et al. Stocking density and rearing environment affect external condition, gonad quantity and gonad grade in onshore sea urchin roe enhancement aquaculture [J]. Aquaculture, 2020, 515 734591-734591.\u003c/li\u003e\n \u003cli\u003eMlingi T F ,Puvanendran V ,Burgerhout E , et al. \u0026ldquo;Influence of short-to-continuous and continuous photoperiods combined with elevated temperatures on sexual maturation in lumpfish (Cyclopterus lumpus, L. 1758)\u0026rdquo; [J]. Aquaculture, 2025, 598 741981-741981.\u003c/li\u003e\n \u003cli\u003eShi M ,Jiang S ,Shi J , et al. Evaluation of Genetic Parameters and Comparison of Stress Tolerance Traits in Different Strains of Litopenaeus vannamei [J]. Animals, 2024, 14 (4):\u003c/li\u003e\n \u003cli\u003eBarralPintos X ,Ar\u0026eacute;valo M ,Escalante K , et al. Sperm quality of Litopenaeus vannamei fed fresh or experimental food in two culture systems [J]. Journal of the World Aquaculture Society, 2023, 55 (2):\u003c/li\u003e\n \u003cli\u003eShuzhan F ,Zheng C ,Yu X , et al. Effects of dietary arachidonic acid on reproduction performance, tissue fatty acid profile and gonadal steroidogenesis in female yellow catfish Pelteobagrus fulvidraco [J]. Aquaculture Nutrition, 2020, 27 (3): 700-711.\u003c/li\u003e\n \u003cli\u003ePamungkas W ,Jusadi D ,Jr Z M , et al. Effect of dietary essential fatty acids on level of oestradiol‐17\u0026beta; and vitellogenin, reproductive performance and larval quality of striped catfish (Pangasianodon hypophthalmus) in out‐of‐spawning season [J]. Aquaculture Research, 2020, 51 (9): 3900-3909.\u003c/li\u003e\n \u003cli\u003eCallan K C ,Laidley W C ,Kling J L , et al. The effects of dietary HUFA level on flame angelfish ( C entropyge loriculus ) spawning, egg quality and early larval characteristics [J]. Aquaculture Research, 2014, 45 (7): 1176-1186.\u003c/li\u003e\n \u003cli\u003eBonvini E ,Parma L ,Mandrioli L , et al. Feeding common sole ( Solea solea ) juveniles with increasing dietary lipid levels affects growth, feed utilization and gut health [J]. Aquaculture, 2015, 449 87-93.\u003c/li\u003e\n \u003cli\u003eCardona E ,Lorgeoux B ,Chim L , et al. Biofloc contribution to antioxidant defence status, lipid nutrition and reproductive performance of broodstock of the shrimp Litopenaeus stylirostris: Consequences for the quality of eggs and larvae [J]. Aquaculture, 2016, 452 252-262.\u003c/li\u003e\n \u003cli\u003eAmran M A ,Ariffin H ,Rahim A A , et al. The effect of lipid level on the growth and reproductive performance of female orange mud crab, Scylla olivacea (Herbst, 1796), during the fattening period [J]. Aquaculture Nutrition, 2021, 27 (6): 2497-2513.\u003c/li\u003e\n \u003cli\u003eWu J ,Liu Y ,Wang Y , et al. The Influence of Dietary n-3 Highly Unsaturated Fatty Acids on Growth, Fatty Acid Profile, Lipid Metabolism, Inflammatory Response, and Intestinal Microflora in F2\u0026nbsp;Generation Female Yangtze Sturgeon (Acipenser dabryanus) [J]. Animals, 2024, 14 (23): 3523-3523.\u003c/li\u003e\n \u003cli\u003eDuc H P ,A.B. M S ,Arifur M R , et al. Effects of n-3 HUFA-enriched Artemia on growth, biochemical response, skeletal morphology and stress resistance of Asian sea bass (Lates calcarifer) larvae reared at high temperature [J]. Aquaculture, 2023, 574\u003c/li\u003e\n \u003cli\u003eHideaki M ,Shinji S ,Mayumi W , et al. Application of genetic disruption of a Nannochloropsis oceanica cell wall synthesizing gene to n-3 HUFA enrichment of Brachionus plicatilis [J]. Aquaculture, 2022, 552\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cuttlefish Pharaoh cuttlefish, wild-caught broodstocks, reproductive performance, hatching rate","lastPublishedDoi":"10.21203/rs.3.rs-8293058/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8293058/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Pharaoh cuttlefish （(\u003cem\u003eSepia pharaonis\u003c/em\u003e） is an important species of cuttlefish in China. Known for its rapid growth rate, strong disease resistance, and high nutritional value, it is considered a highly promising species for aquaculture. During the seed production process, it has been preliminarily observed that wild-caught broodstock exhibit superior reproductive performance compared to the first-generation (F1) artificially cultured broodstock. However, replacing wild-caught broodstock with artificially cultured broodstock for seed production is an inevitable trend and a necessary path for industry upgrading. By conducting in-depth research on the reproductive performance of wild and artificially cultured F1 generation Pharaoh cuttlefish broodstock, this study explores the aspects (spawning quantity and quality) and extent of differences in reproductive performance between them, providing a scientific theoretical basis for the development of the Pharaoh cuttlefish industry and promoting its growth. To clarify the differences in reproductive performance and offspring quality between wild and artificially cultured F1 generation Pharaoh cuttlefish broodstock, a controlled indoor experiment was conducted to compare their spawning quantity and quality, as well as the quality of newly hatched juvenile cuttlefish. Biochemical analyses were also performed to determine the composition and content of amino acids and fatty acids in the fertilized eggs produced by both groups. The results showed that the average number of eggs laid by wild broodstock was 2,050, while that of F1 artificially cultured broodstock was 1,306, indicating that wild broodstock produced 56.97% more eggs than F1 artificially cultured broodstock. The average fertilization rate of eggs produced by wild cuttlefish was 90.23%, whereas that of eggs produced by F1 artificially cultured broodstock was 54.05%, representing a 40.09% decrease compared to wild broodstock. The average long and short diameters of fertilized eggs produced by wild broodstock were 29.55 mm and 16.95 mm, respectively, while those produced by F1 artificially cultured broodstock were 23.15 mm and 16.17 mm, respectively, representing a 27.65% decrease compared to wild broodstock. The average malformation rate of newly hatched juvenile cuttlefish from fertilized eggs produced by wild broodstock was 9.86%, while that of F1 artificially cultured broodstock was 20.40%, representing a 51.76% increase compared to wild broodstock. The average survival rate of juvenile cuttlefish 30 days after hatching from fertilized eggs produced by wild broodstock was 90.54%, while that of F1 artificially cultured broodstock was 81.25%, representing a 10.26% decrease compared to wild broodstock. Biochemical analyses of amino acids and fatty acids revealed that the content of several essential amino acids (such as Lyc, Met, The, Phe) and polyunsaturated fatty acids (such as C20:4n-6, C20:5n-3, and C22:6n-3) in fertilized eggs produced by F1 artificially cultured broodstock was significantly lower than in those produced by wild broodstock (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e), indicating that these nutrients play an important role in the quantity and quality of eggs produced by F1 generation Pharaoh cuttlefish broodstock and the quality of hatched juvenile cuttlefish. In summary, wild Pharaoh cuttlefish broodstock outperform artificially cultured F1 generation broodstock in terms of spawning quantity, quality, and offspring quality. The differences in amino acids and fatty acids in the fertilized eggs produced by the two groups suggest that the quantity and quality of eggs produced by artificially cultured Pharaoh cuttlefish can be improved by optimizing the nutrition of F1 generation broodstock (e.g., by feeding diets rich in Lyc, Met, The, Phe, C18:2n-6, C20:4n-6, C20:5n-3, and C22:6n-3).\u003c/p\u003e","manuscriptTitle":"Comparative analysis of the reproductive performance and offspring quality of pond-reared and wild-caught pharaoh cuttlefish, Sepia pharaonis broodstock","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 14:10:17","doi":"10.21203/rs.3.rs-8293058/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-08T19:31:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-08T16:41:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-08T02:02:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Aquaculture International","date":"2025-12-06T07:58:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"23e78ade-4dd5-40b2-a1ad-23726b7406fd","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T23:08:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-13 14:10:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8293058","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8293058","identity":"rs-8293058","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}