Combined feeding frequency and ration size effects on juvenile Litopenaeus vannamei performance fed diets supplemented with fish hydrolysates

Combined feeding frequency and ration size effects on juvenile Litopenaeus vannamei performance fed diets supplemented with fish hydrolysates | 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 Combined feeding frequency and ration size effects on juvenile Litopenaeus vannamei performance fed diets supplemented with fish hydrolysates Manuel Espinoza-Ortega, César Molina-Poveda, Miguel Jover-Cerdá, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6251899/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Optimum feed ratio and frequency ensure maximum growth and efficient feed utilization in all feeding management strategies. Thus, the present study evaluated the effects of feeding frequency and ration restriction on juveniles of Litopenaeus vannamei (0.8 ± 0.06 g) fed two diets over 53 days. Feeding frequency included two (10:00, 16:00) and four times a day (10:00 h, 16:00, 22:00, 04:00 h), using isonitrogenous diets (35% protein) formulated with fish hydrolysates produced via external (FHEE) or internal (FHIE) enzymes. Feed was supplied at 100% and 80% of apparent satiation. At the end of the experiment, survival was not different among treatments (p > 0.05). Shrimp fed twice showed a significantly higher weight gain than those fed four times (6.27 ± 0.42 vs. 5.76 ± 0.38 g, respectively); feed conversion ratio (FCR) was also higher (1.86 ± 0.16 vs 1.71 ± 0.18, respectively) but not significantly different (p > 0.05). The results demonstrated an improved feed efficiency at 80% compared with 100% satiation (FCR = 1.64 ± 0.07 vs 1.93 ± 0.12, respectively), which was achieved at growth expense (5.79 ± 0.31 vs 6.23 ± 0.51, respectively). No differences in weight gain were observed when comparing the distinct types of diets (FHEE or FHIE). The primary outcomes of the present study indicate a detrimental effect on reduced ration size growth at 80% without any benefits of increasing feeding frequency at night hours. The results of the present study also highlight the impact of apparent satiation and daylight feeding schedules in juvenile L. vannamei . Feeding frequency feeding strategies hydrolysates Litopenaeus vannamei feed restriction Figures Figure 1 Figure 2 1. Introduction The Pacific white shrimp ( Litopenaeus vannamei ) is the most valued species in aquaculture worldwide, representing 30.6 billion dollars, a 51.7% of global crustacean production (FAO, 2022) and 76% of shrimps produced by aquafarming. In Ecuador, shrimp exports reached 1.06 million MT in 2022, while it increased to 1.21 million MT in 2023 (CNA, 2023). Feed is one of the most important factors affecting aquaculture profits and represents 40–60% of the production costs (Tacon et al. 2013 ; Tan Mai et al. 2005). Since feed represents these high production cost proportions, proper feed management is critical for maximizing profits through achieving feed efficiency optimization, enhanced growth, as well as environmental impacts of shrimp farming (Weldon et al. 2021 ). Feeding frequency strategies are critical in determining production cost-effectiveness (Arnold et al. 2016 ). However, feed partitioning is a contradictory subject to date with some authors suggesting that multiple feedings may not be advantageous or have no effect on performance (Smith, Burford & Tabrett 2002 ; Velasco, Lawrence & Castille 1999 ); others indicated that higher feeding frequency may improve growth and enhance feed utilization (Aalimahmoudi, Reyshahri & Bavarsad 2016 ; Napaumpaiporn, Churchid & Taparhudee 2013; Nunes et al. 2019 ). Ahead of the contradictory information about feeding frequency, the effects of other variables have not been addressed to date, such as ration size or formula composition that impact this strategy. Additionally, studies reporting interactions between feeding frequency and diet restriction are limited. Arnold et al. ( 2016 ) described the benefits of a restricted ration on feed efficiency P. monodon juveniles, while Carvalho and Nunes ( 2006 ) reported the possibility of moderately reducing daily feeding rates without affecting performance in juvenile L vannamei . Nutrition is one of the main factors affecting growth, survival and feed efficiency. A growing interest has raised in recent years on hydrolysate inclusion in L. vannamei formulations and in the role of peptides in animal nutrition. The different hydrolysis processes that include chemical, enzymatic or microbial is an attractive form of generating high-quality peptides of suitable size that have physiological and nutritional functions in crustaceans (Hou, Wu, Dai, Wang & Wu 2017 ). Despite hydrolysate potential as chemo-stimulants to promote increased ingestion and thereby affect survival and growth (Carr, 1988 ) limited information is available regarding their effects on L. vannamei performance (Nunes, Carvalho & Sabry-Neto 2006). Some marine ingredients (squid, tuna or crustaceans) can generate a high level of low molecular weight nitrogen components when hydrolyzed under the right conditions, which are highly palatable and contain bioactive and functional properties that improve performance. The enzymatic hydrolysis of marine product residues is normally carried out under conditions that yield final high functionality products (Kristinsson and Rasco, 2000 ). In this sense, some authors have reported L. vannamei growth improvements when fish hydrolysates were used (Córdova-Murueta and García-Carreño, 2002 ; Forster, Babbitt & Smiley 2004 ; Forster, et al. 2011 ; Nguyen, Pérez-Gálvez & Bergé 2012 ). This result is attributed to a better absorption efficiency of hydrolysate and the presence of hydrolysis products, such as amino acids and low molecular weight compounds that enhance attractability and, consequently, intake increase (Aksnes, Hope, Jönsson, Björnsson & Albrektsen 2006 ; Berge and Storebakken, 1996 ). More recently, several studies have demonstrated that hydrolysates improve feeding efficiency and can contribute to better feed conversion ratios, a critical factor in shrimp nutrition (Bøgwald, Herrig, Pedersen, Wubshet, & Eilertsen, 2024 ; Hlordzi et al. 2022 ) Chemoattractants play a crucial role in the formulation of shrimp feeds. These compounds, derived from marine ingredients such as fish hydrolysates, contain bioactive peptides and amino acids that increase palatability and feed consumption, which is essential to maximize growth and feed efficiency (Carr, 1988 ; Aksnes et al. 2006 ). Dietary inclusion of chemoattractants can improve shrimp performance and optimize the cost-benefit ratio, reducing losses due to unconsumed feed and its environmental impact. However, the literature on the specific effects of different types of fish hydrolysates on L. vannamei remains limited, especially in combination with feeding management strategies and ration restriction. Therefore, the present research aimed to evaluate feeding frequency and ration size effects on the Pacific white shrimp ( L. vannamei ) growth, survival, and feed conversion in diets supplemented with two marine chemoattractants. 2. Materials and Methods 2.1. Experimental design The experimental design was a three-way factorial design with two diets, two feeding frequencies and two feeding ratios for a total of eight treatments with three replicates each. Two diets were formulated with different hydrolysates: fish hydrolysate produced via external enzymes (FHEE, pH = 4.36 ± 0.68 ) with 67% of peptides < 1000 Daltons (Da), and fish hydrolysate produced via natural internal enzymes (FHIE, pH = 3.87 ± 0.12) with 88% of peptides with a molecular size < 1000 Da (Fig. 1 ). A factorial array was applied to feeding frequency and ration, which included two feeding frequencies— two times and four times per day—and two feeding rations—100% and 80% satiation. 2.2 Experimental system Shrimp (n = 5000) were collected from a grow-out pond (GranMar, Baja California Sur, MX) and transported to CIBNOR (Centro de Investigaciones Biológicas del Noroeste, La Paz, Baja California Sur, México), where they were stocked in an indoor facility with 18 holding tanks: three 1300-L rectangular tanks (140 x 240 x 50 cm) and fifteen 700-L circular tanks (ø = 94 cm). Before stocking, shrimp were acclimated to laboratory conditions (temperature 27 ± 0.5°C, salinity 38 ± 1 g/L and dissolved oxygen > 5 mg/L) and held for one week in these tanks supplied with filtered seawater (50% water replacement daily). Shrimp were fed a commercial diet with 35% protein content twice daily until the organisms reached approximately 0.8 g. Immediately prior to stocking, 100 shrimp were randomly selected and individually weighed to the nearest 0.01 g (Ohaus Scout® Pro Balance, USA) to estimate the mean and standard deviation (SD). The experimental system consisted of continuous water circulation within 24 fiberglass aquaria with 50 L (50 x 34 x 38 cm) water volume; each tank was equipped with a 250-W submersible heater, air-stone, and seawater filtered supply through a sand filter, cartridge filter (10 µm), and UV light. Water was renewed every day at a rate of 75%. A photoperiod of 12:12-h light/dark cycle (photophase 07:30 to 19:30 h) was maintained throughout the experiment using 28-W tubes for ~ 490 Lux intensity. Shrimp were weighed individually, selected according to required weight based on experimental design and randomly stocked (10 shrimp/aquarium; 47 shrimp/m 2 ) in each 50-L aquarium. The global variation coefficient (VC) was under 10%, and the mean ± SD across all aquaria was 0.8 ± 0.01 g. 2.3 Diets Two pelletized diets were prepared at a laboratory scale and formulated to fulfill or exceed the National Research Council (NRC) requirements for shrimp and consistent with standard industry diets used in Ecuador. The diets contained fish hydrolysates at 2% dietary inclusion level (Table 1 a). The hydrolysate (FHEE) was manufactured from fish mix included: Katsuwonus pelamis, Scomber japonicus, Opisthonema spp. Etrumeus teres, Cetengraulis mysticetus, Auxis spp and Engraulis ringens . The hydrolysate (FHIE) was obtained from fish ( Salmo salar ). The composition of hydrolysates is detailed in Table 2 . Table 1 Ingredients and proximate composition of the experimental diets (g/100g as fed basis). FHEE 1 FHIE 2 Ingredients 3 Vegetable sourced meal 71.04 71.04 Marine animal products 10.00 10.00 Rendered animal by-products 7.45 7.45 Fish hydrolysate 2.00 2.00 Fish oil 1.74 1.74 CaCO 3 , Ca(H₂PO₄)₂, NaCl 3.99 3.99 Lecithin Mix 1.29 1.29 Water 1.21 1.21 Binder 0.52 0.52 Antifungal and mycotoxin sequestrant 0.20 0.20 Vitamin-Mineral Premix 0.56 0.56 Proximate composition Dry matter (g/100g as fed) 89.83 89.30 Protein 35.43 35.20 Ether extract 5.70 5.90 Ash 10.30 10.70 Nitrogen-free extract 17.80 17.90 1 FHEE : Hydrolysate produced by controlled enzymatic hydrolysis of the fresh fish by-products via external enzymes. 2 FHIE : Enzymatically produced, hydrolyzed fish protein with fish produced via natural fish enzymes. 3 The complete formulation is not provided due to proprietary nature of the formulations. Prior to preparing the experimental diets, all macro-ingredients were ground in a laboratory mill (Pavan®, Italy) and passed through a 0.25 mm mesh sieve. The micro-ingredients were mixed in a plastic container, and then added to the macro-ingredients. The dry ingredients of each diet were mixed thoroughly in a food mixer (Kitchen Aid®, USA) before soy lecithin was added. After the soy lecithin was dispersed, water was added (approximately 30% of the total "as is" ingredient weight) and finally mixed. The resulting mixture was pressure-processed in a meat grinder (Torrey® México) through a die with 2-mm diameter holes, as described by (Civera & Guillaume, 1989 ). Pellets were dried to a moisture content of 8–10% in a forced-air oven at 60°C for approximately 8 h and stored at 4°C until used. Table 2 Proximate composition of hydrolysates FHEE and FHIE (g/100g as fed basis). Diet Moisture (%) pH Protein (%) FHEE 59.93 ± 4.83 4.36 ± 0.68 19.80 ± 3.41 FHIE 56.45 ± 0.07 3.87 ± 0.12 31.10 ± 0.71 2.4 Experimental conditions During the 53 days of the trial, oxygen and temperature were measured once every two days, pH and salinity were measured and recorded once a week, and ammonia and nitrites were measured once every ten days. A 100% feeding ration treatment was administered slightly in excess of expected satiety and 80% was calculated based on 100%. Uneaten feed and feces were removed in all treatments by siphoning each tank daily at 08:00 h. The amount of uneaten feed was scored by means of counting the number of pellets and multiplying by average pellet weight; this was used to adjust the following day’s ration accordingly. Within each treatment the following array was applied: two feeding times/day (10:00 h, 16:00 h) and four feeding times/day (10:00 h, 16:00 h, 22:00 h, 04:00 h). The whole daily ration to be administered was weighed and divided manually into similar amounts by volume. All feed rations were distributed in uniform portions and the amount of feed allotted to each aquarium was recorded. The first two rations were administered manually, while the following rations in each treatment group were fed using Fish Mate® F14 Automatic feeders (Pet Mate®, Surrey, England). At the beginning of the day, the automatic feeders were visually checked, and any feed remaining was recorded. The number of shrimp was recorded daily in each aquarium, which was aerated with a single air diffuser. Dissolved oxygen and temperature were monitored daily. Salinity and pH were monitored once a week in each tank. At the beginning of the experiment, feed was supplied at approximately 8% of the biomass, ensuring a marginal excess in 100% treatments. At the end of the trial, feed supply was 5.9% and 4.8% of biomass for 100% and 80% rations, respectively. Ammonia, nitrate, and nitrites in water were analyzed by a chemical colorimetric kit (Mars Fishcare North America, Chalfont, PA, USA) every 15 days. 2.5 Calculations Growth performance and L. vannamei survival for all the groups were calculated with the following equations: Survival (%) = final number of shrimp / initial number of shrimps × 100 Weight gain (g) = (Wf - Wi) Feed conversion ratio (FCR) = Tf / (Wf - Wi) Final biomass (g) = final number of shrimp × Wf Where: Wf represents the final body weight (g), Wi is the initial body weight (g), Tf represents total feed consumption (g) 2.6 Statistical analyses Statistical analyses were performed using Statgraphics® Centurion™ XVII© (Copyright 1982–2014 Statpoint Technologies, Inc). Growth, survival and feed efficiency were analyzed by means of one-way analysis of variance (ANOVA); final weight, final biomass, survival, FCR, growth rate, weight gain, and total consumed feed were analyzed by three-way ANOVA. The model included diet, frequency, and ration size as the main effects and the corresponding interactions. Subsequently, a t-test was applied to find significant differences within each treatment. 3. Results 3.1 Water quality Averages of water quality parameters are shown in Table 3 . Water temperature in all treatments was constant without any significant change (27 ± 0.06 ºC), also salinity (37 ± 0.28 g/L) and dissolved oxygen (5.81 ± 0.08 mg/L) had no significant variations. Ammonia, nitrate and nitrites did not surpass the limits declared in (Boyd, 2016 ). Table 3 Mean (± SD) minimum and maximum values of water quality parameters throughout juvenile shrimp Litopenaeus vannamei (0.8 g initial weight) rearing period stocked at 47 shrimp/m 2 and grown for 53 days in a clear water system. Water parameters Accepted Range Mean Minimum Maximum Temperature (ºC) 18–33 27.05 ± 0.06 26.91 27.14 pH 6–9' 7.97 ± 0.03 7.9 8.08 Dissolved Oxygen (mg/L) 2.5–10 5.81 ± 0.08 5.65 5.98 Salinity (g/L) 1–50¨ 37.28 ± 0.28 36.83 37.62 Ammonia (NH 3 /NH 4 + , mg/L) 0.1-1.0 0.44 ± 0.10 0.3 0.6 Nitrate (NO 3 − , mg/L) 0.2–10 0.61 ± 0.49 0 1 Nitrite (NO 2 − , mg/L) < 1.0 0.1 ± 0.00 0.1 0.1 SD = standard deviation 3.2 Shrimp performance The effect of the type of attractant, ration size, and feeding frequency are presented in Tables 4 and 5 . A three-way ANOVA analysis revealed a significant main effect ( p 0.05) were detected between diet x frequency x ration in any of the zootechnical parameters. Table 4 Three-way analysis of Litopenaeus vannamei (0.8 g initial weight) variance of diet x frequency x ration effects on weight gain of stocked at 47 shrimp/m 2 and grown for 53 days in a clear water system. Sum sq. df Mean Square F value p-value A: Diet 0.0611 1 0.0611 0.5300 0.4753 B: Frequency 1.0621 1 1.0621 9.2800 0.0077 C: Ration 0.8489 1 0.8489 7.4200 0.0150 Diet*Frequency 0.0003 1 0.0003 0.0000 0.9594 Diet*Ration 0.0089 1 0.0089 0.0800 0.7843 Frequency*Ration 0.0920 1 0.0920 0.8000 0.3831 Diet*Frequency*Ration 0.0636 1 0.0636 0.5600 0.4668 Residuals 1.831 16 0.114 Table 5 Three-way analysis of variance of Litopenaeus vannamei (0.8 g initial weight) diet x frequency x ration effects on the feed conversion ratio stocked at 47 shrimp/m 2 and grown for 53 days in a clear water system. Sum sq. df Mean Square F value p-value A: Diet 0.0180 1 0.0180 2.0100 0.1752 B: Frequency 0.1271 1 0.1271 14.2500 0.0017 C: Ration 0.4896 1 0.4896 54.8900 0.0000 Diet*Frequency 0.0284 1 0.0284 3.1800 0.0933 Diet*Ration 0.0000 1 0.0000 0.0000 0.9869 Frequency*Ration 0.0066 1 0.0066 0.7400 0.4033 Diet*Frequency*Ration 0.0186 1 0.0186 2.0800 0.1685 Residuals 0.1427 16 0.0089 After 53 days, survival was high and showed an average of 95 ± 7% (ranging from 87 ± 12% to 100 ± 0%), and no significant differences ( p > 0.05) among treatments were detected (Table 6 ). Mean shrimp final weight of each treatment ranged from 6.38 ± 0.13 to 7.17 ± 0.23 g (Table 6 ), and mean weight gain ranged from 5.58 ± 0.13 g to 6.41 ± 0.23 g per week (Table 6 ). Regarding FCR, the trend revealed that 80% restriction treatments had a tendency towards a better feed efficiency range (1.53 ± 0.08 to 1.77 ± 0.10), while the 100% treatments showed higher values (from 1.83 ± 0.04 to 2.07 ± 0.13). As expected, a higher FCR was observed in the treatments in which the diet was offered at 100%, with the highest consumed feed observed in FHIE treatment at 2 doses and at 100% ration size (130 ± 1.47g), while the lowest consumption was verified with both FHEE and FHIE treatments at 4 doses and 80% satiety (84 ± 1.55g and 86 ± 1.49 g). Table 6 Mean ± standard deviation (SD) of Litopenaeus vannamei growth, survival, and feed efficiency after 53 days of rearing fed two different diets at two feeding frequencies and two ration levels. Diet FHEE FHIE Frequency 2 4 2 4 Ration size 100% 80% 100% 80% 100% 80% 100% 80% Initial weight (g/shrimp) 0.80 ± 0.00 0.80 ± 0.00 0.80 ± 0.01 0.80 ± 0.001 0.80 ± 0.01 0.80 ± 0.00 0.80 ± 0.00 0.80 ± 0.00 Final weight (g/shrimp) 7.17 ± 0.23 6.61 ± 0.18 6.52 ± 0.33 6.41 ± 0.37 7.21 ± 0.30 6.77 ± 0.35 6.77 ± 0.56 6.38 ± 0.13 Final biomass (g) 66.87 ± 1.41 ab 61.68 ± 2.85 ab 56.43 ± 6. 25 a 64.08 ± 3.69 ab 69.67 ± 5.56 b 67.70 ± 3.50 ab 63.14 ± 4.94 ab 61.70 ± 3.99 ab Weight gain (g) 6.38 ± 0.32 5.81 ± 0.17 5.72 ± 0.33 5.61 ± 0.36 6.41 ± 0.29 5.97 ± 0.35 5.97 ± 0.55 5.57 ± 0.13 Consumed feed (g) 116.77 ± 4.11 ab 95.39 ± 1.89 cd 101.98 ± 12.96 bcd 83.83 ± 1.55 d 130.38 ± 1.47 a 105.27 ± 1.38 abc 106.05 ± 9.96 abc 86.33 ± 1.49 d FCR 1.89 ± 0.05 cd 1.70 ± 0.03 abc 1.91 ± 0.16 cd 1.53 ± 0.09 a 2.07 ± 0.13 d 1.77 ± 0.10 abc 1.83 ± 0.04 bcd 1.57 ± 0.06 ab Survival (%) 93 ± 6 93 ± 6 87 ± 12 100 ± 0 97 ± 6 100 ± 0 93 ± 6 97 ± 6 Values ​​followed by different letters in the same row are significantly different ( p < 0.05); SD = standard deviation; FHEE = hydrolized fish with external enzymes; FHIE = hydrolized fish with natural internal enzymes; FCR = feed conversion rate 3.3 Feeding frequency and ration size Average weight gain was statistically greater in the treatments with a 100% ration ( p < 0.05) compared to 80% ration size (Table 7 ); FCR was also the highest when no restriction was applied ( p < 0.05) (Table 8 ). When the organisms were fed twice per day, average weight gain was significantly greater ( p < 0.05) than that observed when diet was distributed four times per day (6.27 ± 0.42 vs 5.76 ± 0.38) ( p < 0.05). Additionally, a significant average weight increment ( p < 0.05) was observed when ration increased from 80 (5.79 ± 0.31) to 100% (6.23 ± 0.51) with a consistent effect for both diets (Table 6 ). When feeding frequency was analyzed for each feeding ration, weight gain (g) was the highest for two feedings (6.40 ± 0.28 vs. 5.85 ± 0.43), but no significant difference appeared for 80%. Table 7 Mean (± SD) of juvenile Litopenaeus vannamei weight gain (g/shrimp) combined by frequencies (2 and 4 feedings) and ration sizes (80 and 100%) after 53 days of rearing fed two different diets at two frequencies and two satiation levels. Ration Frequency 100% 80% Frequency mean Combined 2 feedings 6.40 ± 0.28 Aa 5.89 ± 0.26 B 6.27 ± 0.43 a 4 feedings 5.85 ± 0.43 Ab 5.59 ± 0.25 B 5.76 ± 0.38 b Ration mean 6.23 ± 0.51 A 5.79 ± 0.31 B 6.01 ± 0.36 Values are presented as means ± standard deviation (SD) of three replicates for each feed-frequency-ration size combination. The frequency averages with different superscripts (uppercase per row and lowercase per column) are significantly different (p < 0.05). A significant effect ( p < 0.05) of frequency and ration size on FCR was observed (Table 8 ) after 53 days of testing. Ration size was reduced to 80%, resulting in a statistically lower average FCR than 100% (1.64 ± 0.07 vs. 1.93 ± 0.12, respectively). Feed conversion ratio decreased from 1.86 ± 0.16 when average feeding frequency was considered when the organisms were fed twice a day and 1.71 ± 0.18 when fed four times a day (Table 8 ); FCR decreased from 1.73 ± 0.08 when two doses were fed and at 80% to 1.55 ± 0.07 when fed four doses (Table 8 ). However, this pattern was not the same at 100% of the ration, where FCR (1.99 ± 0.13) was not significantly different ( p > 0.05) with two times a day from that obtained with four times a day (1.87 ± 0.11). Table 8 Mean (± SD) feed conversion rate combined by frequencies (2 and 4 feedings) and ration sizes (80 and 100%) after 53 d of rearing juvenile Litopenaeus vannamei fed two different diets at two feeding frequencies and two satiation levels. Ration Frequency 100% 80% Frequency mean Combined 2 feeds 1.99 ± 0.13 A 1.73 ± 0.08 a B 1.86 ± 0.16 4 feeds 1.87 ± 0.11 A 1.55 ± 0.07 b B 1.71 ± 0.18 Ration mean 1.93 ± 0.12 A 1.64 ± 0.07 B 1.78 ± 0.19 Values for each diet-frequency-ration combination are means (± standard deviation (SD) of three replicates. The frequency averages with different superscripts (uppercase per row and lowercase per column) are significantly different ( p < 0.05). Considering the average for two frequencies, reduction in ration size from 100 to 80% led to a lower combined FCR (1.93 ± 0.12 vs 1.64 ± 0.07). The same pattern was also observed where FCR at 100% (1.99 ± 0.13) was higher at two times per day than at 80% satiation (1.73 ± 0.08) at four times per day when the ration decreased from 100 (1.87 ± 0.11) to 80% satiation (1.55 ± 0.07), which led also to a significant difference in FCR ( p 0.05) were observed in growth, feed conversion and survival between shrimp fed with the different marine hydrolysates. Nevertheless, the consumed FHIE was significantly higher than FHEE diet (107.01 ± 1.76 vs 99.99 ± 1.76, respectively), mainly in the two-meal frequency (Fig. 2 ). Additionally, regarding consumed feed, significant differences were observed ( p < 0.05) between FHIE at 2-dose treatment and 100% of ration size, compared to all other treatments at 80% satiety and even compared to FHEE at 4-dose treatment and 100% ration size. The only exception was registered when FHIE at 2 doses and 100% was compared with FHIE at 2 doses and 80% satiety, where no significant differences were observed ( p > 0,05 ), even though this treatment had a restriction of 20%. 4. Discussion The present study found significant feeding frequency effects and ration restriction size on juvenile L. vannamei growth and FCR. Feeding shrimp four times per day relative to twice a day significantly improved FCR but did not enhance growth; rather, a significantly lower growth was observed under this study conditions with four doses, that included night feeding, because feed consumption was reduced. These findings reveal the notable impact of feeding and ration size schedules on production efficiency when rearing shrimp, highlighting the need to understand this correlation when planning feed management schemes, as stated by Arnold et al. ( 2016 ). 4.1 Feeding frequency Under the present study conditions, our results clearly show that increasing feeding frequency during night hours does not improve growth performance. Mean weight gain with two feedings per day (6.27 ± 0.43 g) was significantly higher compared to four feedings per day (5.76 ± 0.38 g), both at 100% and 80% ration sizes, as shown in Table 7 . The highest results in weight gain were obtained when feed was administered during daylight hours (10:00 h and 16:00 h), while no additional benefit was observed when part of the ration was distributed during night hours (22:00 h and 04:00 h). Findings indicate that L. vannamei , as observed in studies by Nunes, Goddard & Gesterira (1996) and Reymond and Lagardére ( 1990 ), exhibit different feeding behaviors compared to other penaeid shrimps. While most grooved penaeids are typically active and burrow during nighttime (Hindley, 1975 ), L. vannamei showed to be more active in feeding during daylight hours as reported by Pontes et al. ( 2006 ) and Nunes et al. ( 2019 ). In terms of FCR more feedings per day resulted in more efficient feed utilization by shrimp as informed by Aalimahmoudi et al. ( 2016 ), which is verified in lower feed conversion values. Growth improvement with two feed doses instead of four could be understood based on the enzyme profile activities throughout the day, suggesting an oscillatory pattern with feeding peaks at certain times when a maximum feeding activity exists. In crustaceans, certain biological phenomena have been observed to occur rhythmically around the same time (Casillas-Hernández et al. 2006 ). Circadian rhythms affect enzymatic activity, which depends on several exogenous factors, such as age, size, protein sources, and molting and endogenous causes (such as ontogenics, metabolic rates, circadian rhythms) (Lemos, Ezquerra & García-Carreño 2000; Molina, Cadena & Orellana 2000 ). The results of the present study suggests that circadian rhythms might underlie variations in performance at different frequencies. Casillas-Hernández et al. ( 2006 ) studied juvenile L. vannamei enzymatic profile under continuous feed intake and found fluctuation in proteolytic activities. In an experiment with juvenile L. vannamei , a biphasic circadian rhythm was observed with diurnal and nocturnal enzymatic peak activities at 10:00 and 20:00 h. Moreover, as observed, feeding two hours before these peaks (08:00 and 18:00 h) resulted in a significantly higher growth rate, final size, survival, and biomass. These findings suggest that some periods throughout the day exist in which feeding effort should be concentrated, and specific hours depend on L. vannamei physiological status. The results also confirm synchrony between feeding activity and feed use. The biphasic circadian rhythm might explain why no effect was observed in growth when feeding frequency is increased at night. In the present study, shrimp fed two times per day had higher weight gain than those fed four times per day at 100% of ration size (Table 7 ), which is also in agreement with Pontes, De Lima and Arruda (2008),Haz clic o pulse aquí para escribir texto. who reported that increased temporal space between feeding stimulates searching for and ingesting feed. Moreover, regarding feeding schedule, Pontes and Arruda(2005) studied L. vannamei feeding behavior as a function of artificial light and dark photoperiods and observed feeding time was higher in the light phase while swimming occurred mostly during the dark phase. In the same line, Sick, White and Baptist(1973) reported that ingestion rate was proportional to light intensity and inversely related to the time at which the feed was immersed in water in juvenile Litopenaeus setiferus . Furthermore, Robertson, Lawrence & Castille. (1993) reported that day feedings produce more growth in juvenile L. vannamei held in pond enclosures than those at night feedings. Seemingly, L. vannamei are inherently more active during day than at night. More recently, Reis et al. (2021) reported that in outdoor and indoor systems, findings suggest that L. vannamei does not appear to have a specific feeding schedule preference as long as environmental conditions and overall feeding rates are appropriate. However, it is suggested that night feeding may be less efficient compared to day feeding, mainly due to limitations related to dissolved oxygen. During the night, the biological oxygen demand increases due to feeding activity, while oxygen levels are naturally lower. This leads to a higher risk of oxygen depletion, which may require adjustments such as reduced feed rations or more frequent use of mechanical aerators, which increases electrical costs. In terms of growth, shrimp fed exclusively at night or on a 24-hour schedule showed slightly slower growth and lower average final weights compared to day feeders or under the AQ1 (acoustic, on-demand) feeding system. In brief, an oscillating pattern in feeding habits suggests that an increase in feeding frequency should be based on natural behavior and physiological status with higher activity during daytime hours. Our findings show that growth can be sustained by reducing the feeding frequency if an excess amount of feed is provided to meet the nutritional requirements. However, it is important to note that this approach comes with a trade-off: feeding excess -even marginal- compromises feed efficiency (Arnold et al. 2016 ). In terms of FCR, feeding shrimp four times daily resulted in a more efficient feed utilization (1.71 ± 0.18) compared to feeding twice daily (1.86 ± 0.16), as indicated in Table 8 . These findings align with Aalimahmoudi et al. ( 2016 ), who also observed improved FCR with increased feeding frequencies. However, this improved efficiency did not translate into higher growth under our experimental conditions, suggesting that the benefits of increased feeding frequencies are context-dependent and should be evaluated in combination with environmental and operational factors. 4.2 Ration Size The present study demonstrates a detrimental effect of a restricted ration (20% below satiation) in juvenile L. vannamei , even when feeding frequency increased from two to four times per day, because feed consumption was reduced. Feeding at apparent satiety -as demonstrated in the present study- led to an improved growth, since shrimp can consume feed allowing them to meet their nutritional requirements. Additionally, on demand feeding allows reducing stress associated with limited feeding. In this strategy, shrimp are less likely to compete for feed or exhibit aggressive behavior. Reduced stress levels can contribute to overall better health and disease resistance. On the other hand, ration size reduction to 80% improves feed efficiency, which was consistent for both diets, restrictive feeding offers optimal utilization of feed, although it comes at the expense of overall weight gain. Increased feed conversion ratios observed in the present study, such as growth rate increase, indicates that the feeding input plateau was likely approached (Weldon et al. 2021 ). When the maximum weight gain is reached, an inflection point occurs at 101% of the feeding rate; beyond this point, growth and feed utilization decrease rapidly, resulting in a higher feed conversion ratio and diminished growth gains. Therefore, aiming for maximum gain is counteracted by the elevated FCR (Weldon et al. 2021 ). Our results are consistent with those of Arnold et al. ( 2016 ), who reported similar trends in FCR and growth when ration size was restricted. In this study Arnold et al.(2016) reported FCR decrease when ration reduced from 100% satiation (1.52 ± 0.05) to 80% satiation (1.26 ± 0.04). In agreement with our results, the present study also found a FCR reduction from 1.93 ± 0.12 at 100% satiation to 1.64 ± 0.07 at 80% ration size. The effect of reducing FCR as ration size decreases contrasts with that reported by Venero, Davis & Rouse(2007) where feeding at 100% and 75% led to significantly different yields for the two satiation levels: 6482 kg/ha at 100% versus 5054 kg/ha at 75%, respectively, but similar to FCRs for both treatments. On the other hand, the results obtained in the present study are very similar to those reported by Nunes et al. ( 2018 ), observing that feeding shrimp 4.5% of biomass with 0% restriction against 3.4% of biomass with 25% restriction resulted in improved growth performance with higher but still acceptable feed conversion rates. Nunes et al.(2006) found no differences between apparent satiation versus 25% restriction, which suggests the possibility of moderately reduce daily feeding without detrimental effects on growth. Nevertheless, an adverse effect was observed on growth performance at 20% feed restriction. The findings in the present study suggest that a restricted feeding scheme led to a reduced growth rate by limiting the feed available amount to shrimp. With this scheme, shrimp growth may be compromised. A restrictive feed scheme might create a competitive feeding environment, causing increased stress levels, aggression, and dominance behavior among shrimp. Stress can negatively affect their overall health and well-being. Moreover, restrictive feeding might lead to variations in individual growth rates within a population. Some shrimp may consume more feed than others because of dominant behaviors, leading to size disparities and uneven growth distribution within the group. Restrictive feeding schemes also might not provide all the essential nutrients required for optimal shrimp growth and health which can impact various physiological functions. Efficient feeding practices ensure the right feed amount is provided. However, overfeeding should be avoided, thus, the release of excessive nutrient input in the water is important. By providing the precise feed quantity, farmers are able to minimize nutrients and organic matter release, reducing shrimp farming environmental impact (Boyd et al. 2017 ; Weldon et al. 2021 ). 4.3 Type of diet No significant differences were observed using either fish processed with internal (FHIE) or external enzymes (FHEE) in terms of growth and survival, but hydrolysates may enhance aquafeed palatability even though a variety of studies suggest that peptides with lower molecular weights facilitate easier absorption (Carvalho, Sá, Oliva-Teles & Bergot 2004 ), since the hydrolysis process releases smaller peptides that can stimulate feeding response and improve feed intake. Feed consumption was consistently higher in shrimp fed the FHIE diet, particularly at 100% ration size and two feedings per day (130.38 ± 1.47 g for FHIE vs. 116.77 ± 4.11 g for FHEE). This suggests that the FHIE hydrolysate may enhance feed palatability, even though this did not translate into significant differences in growth or FCR under the present study conditions. Hydrolysates with a suitable peptide distribution in the 500–1000 Da range appear to be effective in increasing feed palatability. Studies have shown that smaller peptides improve absorption efficiency and stimulate feeding responses. For example, Carvalho et al. ( 2004 ) found that low-molecular-weight peptides enhance nutrient utilization in fish larvae. Similarly, Kristinsson and Rasco ( 2000 ) reported that hydrolysates with targeted peptide sizes improve palatability and functional properties. Aksnes et al. ( 2006 ) also demonstrated the role of size-fractionated fish hydrolysates in enhancing feed intake and efficiency in aquaculture diets, and finally, Hou et al. ( 2017 ) further highlighted that peptides within this molecular range are bioactive and functional, which may contribute to their effectiveness as feed attractants. Hydrolysates with a suitable peptide distribution in 500–1000 Da range appear to be effectively increasing feed palatability, even though no effect in growth or survival is observed when FHIE is compared with FHEE. Feed consumption seems to be different for diets with two hydrolysates with an average of 99.5 g for FHEE and 107 g for FHIE, which is manifested in homologous diets, 117 vs 130 (2/100%), 95 vs 105 (2/80%), and 102 vs 106 (4/100%), except for 4/80%, which does not have an effect into growth or feed conversion. Further research is required to evaluate different peptide distributions in hydrolysates, as well as a narrower range of ration levels (100 and 90%) in combination of wider feeding frequency series (2, 4, 8, 16 times a day) followed by confirmation of the findings in commercial scenarios. Optimal feeding strategies are essential to minimize feed wastage while ensuring adequate nutrient intake. Studies have shown that improper feed management can lead to increased production costs and environmental impact due to uneaten feed accumulation. While hydrolysates can represent an additional cost in formulation, their ability to enhance feed intake and nutrient absorption may result in better overall profitability by reducing the total amount of feed required per unit of shrimp biomass produced (Hlordzi et al. 2022 ). Summarizing, the three aspects studied: type of feed, feeding frequency and ration size have significant implications on the biological performance of L. vannamei and the final feed cost. In our study, it was observed that feeding twice a day resulted in higher growth, while four daily rations improved feed efficiency (FCR). From an economic perspective, these differences can translate into variations in management decisions and operational costs, such as feed use and labor in farms. Existing literature suggests that proper management of these variables can reduce costs associated with feed waste and energy used in nocturnal feeding, especially when consumption is lower (Reis et al. 2021). Thus, our recommendations highlight the importance of evaluating the relationship between operational cost and biological performance to design more profitable and sustainable feeding strategies. 5. Conclusion The present study demonstrated that feed efficiency benefits of restricted ration are achieved but with less growth, even if feeding frequency was increased. The main outcomes highlight daylight feeding schedule significance and indicate that L. vannamei shows no beneficial effect of restricting or increasing feeding frequency at night hours, and the positive effects of daylight feeding at hours close to juvenile L. vannamei maximum enzymatic reported activities. The findings in the present research suggest that under laboratory conditions, feeding at higher level provides the most advantageous option for optimizing production performance. Declarations Competing interests: The authors declare no competing interests. Author Contribution Conceptualization: MEO, RCC and MJC. Methodology: MEO, RCC and MJC. Formal analysis: MEO, CMP, RCC, MJC. Investigation: MEO, RCC. Writing - original draft preparation: MEO, RCC and MJC. Writing - review and editing: MJC, RCC, and CMP. Supervision: CMP, RCC, MJC. Acknowledgement The authors are grateful to the Ecuadorian Secretary of Higher Education, Science, Technology and Innovation support for providing the scholarship for Manuel Espinoza (Contract 063-2012). The authors also express their gratitude to Gisis S.A., as well as Saúl Zamora and Sandra de La Paz Reyes from the Aquatic Nutrition laboratory at CIBNOR, Mexico for the technical support during the feeding trial, and Diana Dorantes for English language edition. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Aalimahmoudi, M., Reyshahri, A., & Bavarsad, S. S. (2016). Effects of feeding frequency on growth, feed conversion ratio, survival rate and water quality of white leg shrimp. International Journal of Fisheries and Aquatic Studies , 4 (August 2015), 293–297. https://www.fisheriesjournal.com/archives/2016/vol4issue3/PartD/4-2-88.pdf Aksnes, A., Hope, B., Jönsson, E., Björnsson, B. 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Use of fish hydrolysates and fish meal byproducts of the Alaskan fishing industry in diets for pacific white shrimp Litopenaeus vannamei . North American Journal of Aquaculture , 73 (3), 288–295. https://doi.org/10.1080/15222055.2011.598371 Hindley, J. P. R. (1975). Effects of endogenous and some exogenous factors on the activity of the juvenile banana prawn Penaeus merguiensis . Marine Biology , 29 (1), 1–8. https://doi.org/10.1007/BF00395521 Hlordzi, V., Wang, J., Kuebutornye, F. K. A., Yang, X., Tan, B., Li, T., … Shuyan, C. (2022). Hydrolysed fish protein powder is better at the growth performance, hepatopancreas and intestinal development of Pacific white shrimp (Litopenaeus vannamei). Aquaculture Reports, 23(November 2021), 101025. https://doi.org/10.1016/j.aqrep.2022.101025 Hou, Y., Wu, Z., Dai, Z., Wang, G., & Wu, G. (2017). Protein hydrolysates in animal nutrition: Industrial production, bioactive peptides, and functional significance. Journal of Animal Science and Biotechnology , 8 (1), 1–13. https://doi.org/10.1186/s40104-017-0153-9 Kristinsson, H. G., & Rasco, B. A. (2000). Fish Protein Hydrolysates: Production, Biochemical, and Functional Properties. In Critical Reviews in Food Science and Nutrition (Vol. 40, Issue 1). https://doi.org/10.1080/10408690091189266 Lemos, D., Ezquerra, J. M., & Garcia-Carreño, F. L. (2000). Protein digestion in penaeid shrimp: Digestive proteinases, proteinase inhibitors and feed digestibility. Aquaculture , 186 (1–2), 89–105. https://doi.org/10.1016/S0044-8486(99)00371-3 Molina, C., Cadena, E., & Orellana, F. (2000). Alimentación de camarones en relación a la actividad enzimática como una respuesta natural al ritmo circadiano y ciclo de muda. In: Cruz-Suárez, L.E., Ricque-Marie, D., Tapia-Salazar, M., Olvera-Novoa, M.A. y Civera-Cerecedo. R, (Eds.). Avances en Nutrición Acuícola V. Memorias del V Simposium Internacional de Nutrición Acuícola. 19-22 Noviembre, 2000. Mérida, Yucatán, México. Págs. 358-380 Napaumpaiporn, T., Chuchird, N., & Taparhudee, W. (2013). Study on the Efficiency of Three Different Feeding Techniques in the Culture of Pacific White Shrimp ( Litopenaeus vannamei ). Kasetsart University Fisheries Research Bulletin , 37 (2), 8–16. National Research Council. (2011). Nutrient requirements off ish and shrimp. National Academies Press. Nguyen, H. T. M., Pérez-Gálvez, R., & Bergé, J. P. (2012). Effect of diets containing tuna head hydrolysates on the survival and growth of shrimp Penaeus vannamei . Aquaculture , 324–325 , 127–134. https://doi.org/10.1016/j.aquaculture.2011.11.014 Nunes, A. J. P., Goddard, S., & Gesteira, T. C. V. (1996). Feeding activity patterns of the southern brown shrimp Penaeus subtilis under semi-intensive culture in NE Brazil. Aquaculture , 144 (4), 371–386. https://doi.org/10.1016/0044-8486(96)01297-5 Nunes, A., Sá, M., Carvalho, E., & Sabry Neto, H. (2006). Growth performance of the white shrimp Litopenaeus vannamei reared under time and rate restriction feeding regimes in a controlled culture system. Aquaculture , 253 (1–4), 646–652. https://doi.org/10.1016/j.aquaculture.2005.09.023 Nunes, A., Sabry-Neto, H., Pires da Silva, F. H., Rodrigues de Oliveira-Neto, A. & Masagounder, K. (2019). Multiple feedings enhance the growth performance and feed efficiency of juvenile Litopenaeus vannamei when fed a low-fish meal amino acid-supplemented diet. Aquaculture International , 27 (2), 337–347. https://doi.org/10.1007/s10499-018-0330-7 Nunes, C. R., Da Silva, C., Clovis, P., & Dos Santos, J. (2018). Evaluation of feeding rates in the production of Litopenaeus vannamei shrimp using artificial substrates. Ciencia Animal Brasileira . https://doi.org/https://doi.org/10.1590/1809-6891v19e-50805 Pontes, C. S., & Arruda, M. D. F. (2005). Comportamento de Litopenaeus vannamei (Boone) (Crustacea, Decapoda, Penaeidae) em função da oferta do alimento artificial nas fases clara e escura do período de 24 horas artificial fases clara escura horas. Revista Brasileira de Zoologia , 22 (3), 648–652. Pontes, C. S., Arruda, M. D. F., De Lara Menezes, A. A., & De Lima, P. P. (2006). Daily activity pattern of the marine shrimp Litopenaeus vannamei (Boone 1931) juveniles under laboratory conditions. Aquaculture Research , 37 (10), 1001–1006. https://doi.org/10.1111/j.1365-2109.2006.01519.x Pontes, C. S., De Lima, P. P., & Arruda M de F. (2008). Feeding responses of juvenile shrimp Litopenaeus vannamei (Boone) fed at different frequencies under laboratory conditions. Aquaculture Research , 1416–1422. https://doi.org/10.1111/j.1365-2109.2008.02011.xKotusReis, J., Weldon, A., Ito, P., Stites, W., Rhodes, M., & Davis, D. A. (2021). Automated feeding systems for shrimp: Effects of feeding schedules and passive feedback feeding systems. Aquaculture, 541. https://doi.org/10.1016/j.aquaculture.2021.736800 Reymond, H., & Lagardére, J. P. (1990). Feeding Rhythms and Food of Penaeus japonicus Bate (Crustacea, Penaeidae) in Salt Marsh Ponds: Role of Halophilic Entomofauna. Aquaculture, 84 (1990) 125-143 , 84 , 125–143. Robertson, L., Lawrence, A. L., & Castille, F. (1993). Effect of feeding frequency and feeding time on growth of Penaeus vannamei (Boone). Aquaculture and Fisheries Management 1993, 24, 1-6. Sick, L. V., White, D., & Baptist, G. (1973). The effect of duration of feeding, amount of food, light intensity, and animal size on rate of ingestion of pelleted food by juvenile penaeid shrimp. Progressive Fish-Culturist , 35 (1), 22–26. https://doi.org/10.1577/1548-8659(1973)35[22:TEODOF]2.0.CO;2 Smith, D. M., Burford, M. A., & Tabrett, S. J. (2002). The effect of feeding frequency on water quality and growth of the black tiger shrimp ( Penaeus monodon ). Aquaculture , 207 , 125–136. Tacon, A. G. J., Jory, D., & Nunes, A. (2013). Shrimp feed management: issues and perspectives. FAO Fisheries and Aquaculture Technical , 8 , 481–488. Tan, B., Mai, K., Zheng, S., Zhou, Q., Liu, L., & Yu, Y. (2005). Replacement of fish meal by meat and bone meal in practical diets for the white shrimp Litopenaeus vannamei (Boone). Aquaculture Research , 36 (5), 439–444. https://doi.org/10.1111/j.1365-2109.2005.01223.x Velasco, M., Lawrence, A., & Castille, F. (1999). Effect of variations in daily feeding frequency and ration size on growth of shrimp, Litopenaeus vannamei (Boone), in zero-water exchange culture tanks. Aquaculture , 179(1-4), 141-148 Venero, J. A., Davis, D. A., & Rouse, D. B. (2007). Variable feed allowance with constant protein input for the pacific white shrimp Litopenaeus vannamei reared under semi-intensive conditions in tanks and ponds. Aquaculture , 269 (1–4), 490–503. https://doi.org/10.1016/j.aquaculture.2007.02.055 Weldon, A., Davis, D. A., Rhodes, M., Reis, J., Stites, W., & Ito, P. (2021). Feed management of Litopenaeus vannamei in a high density biofloc system. Aquaculture , 544 (April), 737074. https://doi.org/10.1016/j.aquaculture.2021.737074 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-6251899","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":434590467,"identity":"80129629-0362-4d39-98ca-bd0b1b5d47be","order_by":0,"name":"Manuel Espinoza-Ortega","email":"","orcid":"","institution":"Skretting, Ecuador, Research and Development Program","correspondingAuthor":false,"prefix":"","firstName":"Manuel","middleName":"","lastName":"Espinoza-Ortega","suffix":""},{"id":434590468,"identity":"73af77b8-0238-4497-9ab5-fbe17e6382ff","order_by":1,"name":"César Molina-Poveda","email":"","orcid":"","institution":"Skretting, Ecuador, Research and Development Program","correspondingAuthor":false,"prefix":"","firstName":"César","middleName":"","lastName":"Molina-Poveda","suffix":""},{"id":434590469,"identity":"8e0ad669-7f7b-4694-9c93-121cfa863e31","order_by":2,"name":"Miguel Jover-Cerdá","email":"","orcid":"","institution":"Universitat Politècnica de València, Spain, Research Group of Aquaculture and Biodiversity, Institute of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"","lastName":"Jover-Cerdá","suffix":""},{"id":434590470,"identity":"c8368f60-92dc-4267-b001-184ece8a9d92","order_by":3,"name":"Roberto Civera-Cerecedo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYFACxgYQycPY3sBwmCQtcsw9B4jWAgHG7DMSGJiJUmrefrhN4uMeu8TemY8fHi7cYZPPIHbGAK8WmTOJbZIzniUnzpydZnB45pk0ywbptAS8WiQkGJuNeQ4wJ26cncNwmLftsAGDdPIBwlr+HKhP3H/zDExLYgMhLY2PGQ4cNmacwUOsLTyJjQ97DhyXY+wB+aUtzYCNoF/Yjz848ONANTAqDz/+XNhmY8AvnYM/xDABG4nqR8EoGAWjYBRgAQCnvkYBtFsWeQAAAABJRU5ErkJggg==","orcid":"","institution":"Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mexico, Laboratorio de Nutrición Acuícola","correspondingAuthor":true,"prefix":"","firstName":"Roberto","middleName":"","lastName":"Civera-Cerecedo","suffix":""}],"badges":[],"createdAt":"2025-03-18 10:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6251899/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6251899/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79454359,"identity":"c19434c6-bb7b-4d21-acf8-8fe2fed4f730","added_by":"auto","created_at":"2025-03-28 15:37:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":26428,"visible":true,"origin":"","legend":"\u003cp\u003ePeptide size profile for the two fish hydrolysate external enzymes (\u003cstrong\u003eFHEE\u003c/strong\u003e and natural internal enzyme \u003cstrong\u003eFHIE\u003c/strong\u003e) used in the experimental diets.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6251899/v1/21e3eaa17532e31b7ebeda85.jpg"},{"id":79454360,"identity":"cc04eb44-acce-4639-8bd1-c18ba3fac7c5","added_by":"auto","created_at":"2025-03-28 15:37:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25464,"visible":true,"origin":"","legend":"\u003cp\u003eAttractant effect on consumed feed (g) by shrimp fed two feeding frequencies and rations\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6251899/v1/c20841875448688dd857de96.jpg"},{"id":79987672,"identity":"781c96e6-a0d2-4648-b852-c5291ce0d9f2","added_by":"auto","created_at":"2025-04-06 08:46:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1053170,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6251899/v1/03e24c40-dc72-4ac1-98ad-03b40f36d87c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Combined feeding frequency and ration size effects on juvenile Litopenaeus vannamei performance fed diets supplemented with fish hydrolysates","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe Pacific white shrimp (\u003cem\u003eLitopenaeus vannamei\u003c/em\u003e) is the most valued species in aquaculture worldwide, representing 30.6\u0026nbsp;billion dollars, a 51.7% of global crustacean production (FAO, 2022) and 76% of shrimps produced by aquafarming. In Ecuador, shrimp exports reached 1.06\u0026nbsp;million MT in 2022, while it increased to 1.21\u0026nbsp;million MT in 2023 (CNA, 2023). Feed is one of the most important factors affecting aquaculture profits and represents 40\u0026ndash;60% of the production costs (Tacon et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tan Mai et al. 2005). Since feed represents these high production cost proportions, proper feed management is critical for maximizing profits through achieving feed efficiency optimization, enhanced growth, as well as environmental impacts of shrimp farming (Weldon et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFeeding frequency strategies are critical in determining production cost-effectiveness (Arnold et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, feed partitioning is a contradictory subject to date with some authors suggesting that multiple feedings may not be advantageous or have no effect on performance (Smith, Burford \u0026amp; Tabrett \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Velasco, Lawrence \u0026amp; Castille \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1999\u003c/span\u003e); others indicated that higher feeding frequency may improve growth and enhance feed utilization (Aalimahmoudi, Reyshahri \u0026amp; Bavarsad \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Napaumpaiporn, Churchid \u0026amp; Taparhudee 2013; Nunes et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Ahead of the contradictory information about feeding frequency, the effects of other variables have not been addressed to date, such as ration size or formula composition that impact this strategy.\u003c/p\u003e \u003cp\u003eAdditionally, studies reporting interactions between feeding frequency and diet restriction are limited. Arnold et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) described the benefits of a restricted ration on feed efficiency \u003cem\u003eP. monodon\u003c/em\u003e juveniles, while Carvalho and Nunes (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) reported the possibility of moderately reducing daily feeding rates without affecting performance in juvenile \u003cem\u003eL vannamei\u003c/em\u003e. Nutrition is one of the main factors affecting growth, survival and feed efficiency. A growing interest has raised in recent years on hydrolysate inclusion in \u003cem\u003eL. vannamei\u003c/em\u003e formulations and in the role of peptides in animal nutrition. The different hydrolysis processes that include chemical, enzymatic or microbial is an attractive form of generating high-quality peptides of suitable size that have physiological and nutritional functions in crustaceans (Hou, Wu, Dai, Wang \u0026amp; Wu \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite hydrolysate potential as chemo-stimulants to promote increased ingestion and thereby affect survival and growth (Carr, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) limited information is available regarding their effects on \u003cem\u003eL. vannamei\u003c/em\u003e performance (Nunes, Carvalho \u0026amp; Sabry-Neto 2006). Some marine ingredients (squid, tuna or crustaceans) can generate a high level of low molecular weight nitrogen components when hydrolyzed under the right conditions, which are highly palatable and contain bioactive and functional properties that improve performance. The enzymatic hydrolysis of marine product residues is normally carried out under conditions that yield final high functionality products (Kristinsson and Rasco, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this sense, some authors have reported \u003cem\u003eL. vannamei\u003c/em\u003e growth improvements when fish hydrolysates were used (C\u0026oacute;rdova-Murueta and Garc\u0026iacute;a-Carre\u0026ntilde;o, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Forster, Babbitt \u0026amp; Smiley \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Forster, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Nguyen, P\u0026eacute;rez-G\u0026aacute;lvez \u0026amp; Berg\u0026eacute; \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This result is attributed to a better absorption efficiency of hydrolysate and the presence of hydrolysis products, such as amino acids and low molecular weight compounds that enhance attractability and, consequently, intake increase (Aksnes, Hope, J\u0026ouml;nsson, Bj\u0026ouml;rnsson \u0026amp; Albrektsen \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Berge and Storebakken, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMore recently, several studies have demonstrated that hydrolysates improve feeding efficiency and can contribute to better feed conversion ratios, a critical factor in shrimp nutrition (B\u0026oslash;gwald, Herrig, Pedersen, Wubshet, \u0026amp; Eilertsen, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Hlordzi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eChemoattractants play a crucial role in the formulation of shrimp feeds. These compounds, derived from marine ingredients such as fish hydrolysates, contain bioactive peptides and amino acids that increase palatability and feed consumption, which is essential to maximize growth and feed efficiency (Carr, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Aksnes et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Dietary inclusion of chemoattractants can improve shrimp performance and optimize the cost-benefit ratio, reducing losses due to unconsumed feed and its environmental impact. However, the literature on the specific effects of different types of fish hydrolysates on \u003cem\u003eL. vannamei\u003c/em\u003e remains limited, especially in combination with feeding management strategies and ration restriction.\u003c/p\u003e \u003cp\u003eTherefore, the present research aimed to evaluate feeding frequency and ration size effects on the Pacific white shrimp (\u003cem\u003eL. vannamei\u003c/em\u003e) growth, survival, and feed conversion in diets supplemented with two marine chemoattractants.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Experimental design\u003c/h2\u003e \u003cp\u003eThe experimental design was a three-way factorial design with two diets, two feeding frequencies and two feeding ratios for a total of eight treatments with three replicates each. Two diets were formulated with different hydrolysates: fish hydrolysate produced via external enzymes (FHEE, pH\u0026thinsp;=\u0026thinsp;4.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 ) with 67% of peptides\u0026thinsp;\u0026lt;\u0026thinsp;1000 Daltons (Da), and fish hydrolysate produced via natural internal enzymes (FHIE, pH\u0026thinsp;=\u0026thinsp;3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12) with 88% of peptides with a molecular size\u0026thinsp;\u0026lt;\u0026thinsp;1000 Da (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A factorial array was applied to feeding frequency and ration, which included two feeding frequencies\u0026mdash; two times and four times per day\u0026mdash;and two feeding rations\u0026mdash;100% and 80% satiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental system\u003c/h2\u003e \u003cp\u003eShrimp (n\u0026thinsp;=\u0026thinsp;5000) were collected from a grow-out pond (GranMar, Baja California Sur, MX) and transported to CIBNOR (Centro de Investigaciones Biol\u0026oacute;gicas del Noroeste, La Paz, Baja California Sur, M\u0026eacute;xico), where they were stocked in an indoor facility with 18 holding tanks: three 1300-L rectangular tanks (140 x 240 x 50 cm) and fifteen 700-L circular tanks (\u0026oslash; = 94 cm). Before stocking, shrimp were acclimated to laboratory conditions (temperature 27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C, salinity 38\u0026thinsp;\u0026plusmn;\u0026thinsp;1 g/L and dissolved oxygen\u0026thinsp;\u0026gt;\u0026thinsp;5 mg/L) and held for one week in these tanks supplied with filtered seawater (50% water replacement daily). Shrimp were fed a commercial diet with 35% protein content twice daily until the organisms reached approximately 0.8 g. Immediately prior to stocking, 100 shrimp were randomly selected and individually weighed to the nearest 0.01 g (Ohaus Scout\u0026reg; Pro Balance, USA) to estimate the mean and standard deviation (SD).\u003c/p\u003e \u003cp\u003eThe experimental system consisted of continuous water circulation within 24 fiberglass aquaria with 50 L (50 x 34 x 38 cm) water volume; each tank was equipped with a 250-W submersible heater, air-stone, and seawater filtered supply through a sand filter, cartridge filter (10 \u0026micro;m), and UV light. Water was renewed every day at a rate of 75%. A photoperiod of 12:12-h light/dark cycle (photophase 07:30 to 19:30 h) was maintained throughout the experiment using 28-W tubes for ~\u0026thinsp;490 Lux intensity.\u003c/p\u003e \u003cp\u003eShrimp were weighed individually, selected according to required weight based on experimental design and randomly stocked (10 shrimp/aquarium; 47 shrimp/m\u003csup\u003e2\u003c/sup\u003e) in each 50-L aquarium. The global variation coefficient (VC) was under 10%, and the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD across all aquaria was 0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 g.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Diets\u003c/h2\u003e \u003cp\u003eTwo pelletized diets were prepared at a laboratory scale and formulated to fulfill or exceed the National Research Council (NRC) requirements for shrimp and consistent with standard industry diets used in Ecuador. The diets contained fish hydrolysates at 2% dietary inclusion level (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The hydrolysate (FHEE) was manufactured from fish mix included: \u003cem\u003eKatsuwonus pelamis, Scomber japonicus, Opisthonema spp. Etrumeus teres, Cetengraulis mysticetus, Auxis spp and Engraulis ringens\u003c/em\u003e. The hydrolysate (FHIE) was obtained from fish (\u003cem\u003eSalmo salar\u003c/em\u003e). The composition of hydrolysates is detailed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIngredients and proximate composition of the experimental diets (g/100g as fed basis).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eFHEE\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFHIE\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIngredients\u003c/b\u003e\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVegetable sourced meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e71.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e71.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMarine animal products\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRendered animal by-products\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e7.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e7.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFish hydrolysate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFish oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaCO\u003csub\u003e3\u003c/sub\u003e, Ca(H₂PO₄)₂, NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e3.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e3.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLecithin Mix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBinder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntifungal and mycotoxin sequestrant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVitamin-Mineral Premix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eProximate composition\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDry matter (g/100g as fed)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e89.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e89.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e35.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e35.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEther extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e5.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e5.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e10.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e10.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrogen-free extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e17.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e17.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003e \u003cb\u003eFHEE\u003c/b\u003e: Hydrolysate produced by controlled enzymatic hydrolysis of the fresh fish by-products via external enzymes.\u003c/p\u003e \u003cp\u003e \u003csup\u003e2\u003c/sup\u003e \u003cb\u003eFHIE\u003c/b\u003e: Enzymatically produced, hydrolyzed fish protein with fish produced via natural fish enzymes.\u003c/p\u003e \u003cp\u003e \u003csup\u003e3\u003c/sup\u003e The complete formulation is not provided due to proprietary nature of the formulations.\u003c/p\u003e \u003cp\u003ePrior to preparing the experimental diets, all macro-ingredients were ground in a laboratory mill (Pavan\u0026reg;, Italy) and passed through a 0.25 mm mesh sieve. The micro-ingredients were mixed in a plastic container, and then added to the macro-ingredients. The dry ingredients of each diet were mixed thoroughly in a food mixer (Kitchen Aid\u0026reg;, USA) before soy lecithin was added. After the soy lecithin was dispersed, water was added (approximately 30% of the total \"as is\" ingredient weight) and finally mixed. The resulting mixture was pressure-processed in a meat grinder (Torrey\u0026reg; M\u0026eacute;xico) through a die with 2-mm diameter holes, as described by (Civera \u0026amp; Guillaume, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Pellets were dried to a moisture content of 8\u0026ndash;10% in a forced-air oven at 60\u0026deg;C for approximately 8 h and stored at 4\u0026deg;C until used.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProximate composition of hydrolysates FHEE and FHIE (g/100g as fed basis).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMoisture (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtein (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFHEE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.93\u0026thinsp;\u0026plusmn;\u0026thinsp;4.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.80\u0026thinsp;\u0026plusmn;\u0026thinsp;3.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFHIE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental conditions\u003c/h2\u003e \u003cp\u003eDuring the 53 days of the trial, oxygen and temperature were measured once every two days, pH and salinity were measured and recorded once a week, and ammonia and nitrites were measured once every ten days. A 100% feeding ration treatment was administered slightly in excess of expected satiety and 80% was calculated based on 100%. Uneaten feed and feces were removed in all treatments by siphoning each tank daily at 08:00 h. The amount of uneaten feed was scored by means of counting the number of pellets and multiplying by average pellet weight; this was used to adjust the following day\u0026rsquo;s ration accordingly.\u003c/p\u003e \u003cp\u003eWithin each treatment the following array was applied: two feeding times/day (10:00 h, 16:00 h) and four feeding times/day (10:00 h, 16:00 h, 22:00 h, 04:00 h). The whole daily ration to be administered was weighed and divided manually into similar amounts by volume. All feed rations were distributed in uniform portions and the amount of feed allotted to each aquarium was recorded. The first two rations were administered manually, while the following rations in each treatment group were fed using Fish Mate\u0026reg; F14 Automatic feeders (Pet Mate\u0026reg;, Surrey, England).\u003c/p\u003e \u003cp\u003eAt the beginning of the day, the automatic feeders were visually checked, and any feed remaining was recorded. The number of shrimp was recorded daily in each aquarium, which was aerated with a single air diffuser. Dissolved oxygen and temperature were monitored daily. Salinity and pH were monitored once a week in each tank. At the beginning of the experiment, feed was supplied at approximately 8% of the biomass, ensuring a marginal excess in 100% treatments. At the end of the trial, feed supply was 5.9% and 4.8% of biomass for 100% and 80% rations, respectively.\u003c/p\u003e \u003cp\u003eAmmonia, nitrate, and nitrites in water were analyzed by a chemical colorimetric kit (Mars Fishcare North America, Chalfont, PA, USA) every 15 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Calculations\u003c/h2\u003e \u003cp\u003eGrowth performance and \u003cem\u003eL. vannamei\u003c/em\u003e survival for all the groups were calculated with the following equations:\u003c/p\u003e \u003cp\u003eSurvival (%)\u0026thinsp;=\u0026thinsp;final number of shrimp / initial number of shrimps \u0026times; 100\u003c/p\u003e \u003cp\u003eWeight gain (g) = (Wf - Wi)\u003c/p\u003e \u003cp\u003eFeed conversion ratio (FCR)\u0026thinsp;=\u0026thinsp;Tf / (Wf - Wi)\u003c/p\u003e \u003cp\u003eFinal biomass (g)\u0026thinsp;=\u0026thinsp;final number of shrimp \u0026times; Wf\u003c/p\u003e \u003cp\u003eWhere: Wf represents the final body weight (g), Wi is the initial body weight (g),\u003c/p\u003e \u003cp\u003eTf represents total feed consumption (g)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Statgraphics\u0026reg; Centurion\u0026trade; XVII\u0026copy; (Copyright 1982\u0026ndash;2014 Statpoint Technologies, Inc). Growth, survival and feed efficiency were analyzed by means of one-way analysis of variance (ANOVA); final weight, final biomass, survival, FCR, growth rate, weight gain, and total consumed feed were analyzed by three-way ANOVA. The model included diet, frequency, and ration size as the main effects and the corresponding interactions. Subsequently, a t-test was applied to find significant differences within each treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Water quality\u003c/h2\u003e \u003cp\u003eAverages of water quality parameters are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Water temperature in all treatments was constant without any significant change (27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 \u0026ordm;C), also salinity (37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 g/L) and dissolved oxygen (5.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 mg/L) had no significant variations. Ammonia, nitrate and nitrites did not surpass the limits declared in (Boyd, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean (\u0026plusmn;\u0026thinsp;SD) minimum and maximum values of water quality parameters throughout juvenile shrimp \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e (0.8 g initial weight) rearing period stocked at 47 shrimp/m\u003csup\u003e2\u003c/sup\u003e and grown for 53 days in a clear water system.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAccepted Range\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMinimum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMaximum\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature (\u0026ordm;C)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u0026ndash;33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u0026ndash;9'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDissolved Oxygen (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalinity (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;50\u0026uml;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmmonia (NH\u003csub\u003e3\u003c/sub\u003e/NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1-1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrite (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eSD\u0026thinsp;=\u0026thinsp;standard deviation\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Shrimp performance\u003c/h2\u003e \u003cp\u003eThe effect of the type of attractant, ration size, and feeding frequency are presented in Tables\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. A three-way ANOVA analysis revealed a significant main effect (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for frequency and ration in weight gain, and FCR. However, no significance was observed in survival. No significant interactions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were detected between diet x frequency x ration in any of the zootechnical parameters.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThree-way analysis of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e (0.8 g initial weight) variance of diet x frequency x ration effects on weight gain of stocked at 47 shrimp/m\u003csup\u003e2\u003c/sup\u003e and grown for 53 days in a clear water system.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSum sq.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean Square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ep-value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA: Diet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0611\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0611\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4753\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB: Frequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0621\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0621\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.2800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0077\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC: Ration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8489\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.8489\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.4200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiet*Frequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9594\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiet*Ration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.7843\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFrequency*Ration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0920\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0920\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.8000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.3831\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiet*Frequency*Ration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4668\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResiduals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.831\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThree-way analysis of variance of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e (0.8 g initial weight) diet x frequency x ration effects on the feed conversion ratio stocked at 47 shrimp/m\u003csup\u003e2\u003c/sup\u003e and grown for 53 days in a clear water system.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSum sq.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean Square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ep-value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA: Diet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.0100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1752\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB: Frequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1271\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1271\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.2500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC: Ration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.4896\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4896\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54.8900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiet*Frequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.1800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0933\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiet*Ration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9869\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFrequency*Ration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.7400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4033\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiet*Frequency*Ration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.0800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1685\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResiduals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1427\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAfter 53 days, survival was high and showed an average of 95\u0026thinsp;\u0026plusmn;\u0026thinsp;7% (ranging from 87\u0026thinsp;\u0026plusmn;\u0026thinsp;12% to 100\u0026thinsp;\u0026plusmn;\u0026thinsp;0%), and no significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) among treatments were detected (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Mean shrimp final weight of each treatment ranged from 6.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 to 7.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 g (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), and mean weight gain ranged from 5.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 g to 6.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 g per week (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding FCR, the trend revealed that 80% restriction treatments had a tendency towards a better feed efficiency range (1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 to 1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10), while the 100% treatments showed higher values (from 1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 to 2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13).\u003c/p\u003e \u003cp\u003eAs expected, a higher FCR was observed in the treatments in which the diet was offered at 100%, with the highest consumed feed observed in FHIE treatment at 2 doses and at 100% ration size (130\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47g), while the lowest consumption was verified with both FHEE and FHIE treatments at 4 doses and 80% satiety (84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55g and 86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49 g).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of Litopenaeus vannamei growth, survival, and feed efficiency after 53 days of rearing fed two different diets at two feeding frequencies and two ration levels.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"14\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiet\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFHEE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eFHIE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRation size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eInitial weight (g/shrimp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal weight (g/shrimp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e6.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e6.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal biomass (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.85\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e56.43\u0026thinsp;\u0026plusmn;\u0026thinsp;6. 25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e64.08\u0026thinsp;\u0026plusmn;\u0026thinsp;3.69\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e69.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.56\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e67.70\u0026thinsp;\u0026plusmn;\u0026thinsp;3.50\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e63.14\u0026thinsp;\u0026plusmn;\u0026thinsp;4.94\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e61.70\u0026thinsp;\u0026plusmn;\u0026thinsp;3.99\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight gain (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e5.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e5.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConsumed feed (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e116.77\u0026thinsp;\u0026plusmn;\u0026thinsp;4.11\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e101.98\u0026thinsp;\u0026plusmn;\u0026thinsp;12.96\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e83.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e130.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e105.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e106.05\u0026thinsp;\u0026plusmn;\u0026thinsp;9.96\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e86.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFCR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurvival (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e93\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e87\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e97\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e93\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e97\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"14\"\u003eValues ​​followed by different letters in the same row are significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); SD\u0026thinsp;=\u0026thinsp;standard deviation; FHEE\u0026thinsp;=\u0026thinsp;hydrolized fish with external enzymes; FHIE\u0026thinsp;=\u0026thinsp;hydrolized fish with natural internal enzymes; FCR\u0026thinsp;=\u0026thinsp;feed conversion rate\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Feeding frequency and ration size\u003c/h2\u003e \u003cp\u003eAverage weight gain was statistically greater in the treatments with a 100% ration (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to 80% ration size (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e); FCR was also the highest when no restriction was applied (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). When the organisms were fed twice per day, average weight gain was significantly greater (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than that observed when diet was distributed four times per day (6.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42 vs 5.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, a significant average weight increment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed when ration increased from 80 (5.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31) to 100% (6.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51) with a consistent effect for both diets (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen feeding frequency was analyzed for each feeding ration, weight gain (g) was the highest for two feedings (6.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 vs. 5.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43), but no significant difference appeared for 80%.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean (\u0026plusmn;\u0026thinsp;SD) of juvenile Litopenaeus vannamei weight gain (g/shrimp) combined by frequencies (2 and 4 feedings) and ration sizes (80 and 100%) after 53 days of rearing fed two different diets at two frequencies and two satiation levels.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFrequency mean\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCombined\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 feedings\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 feedings\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003eAb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRation mean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of three replicates for each feed-frequency-ration size combination. The frequency averages with different superscripts (uppercase per row and lowercase per column) are significantly different \u003cem\u003e(p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eA significant effect (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of frequency and ration size on FCR was observed (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) after 53 days of testing. Ration size was reduced to 80%, resulting in a statistically lower average FCR than 100% (1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 vs. 1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, respectively). Feed conversion ratio decreased from 1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 when average feeding frequency was considered when the organisms were fed twice a day and 1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 when fed four times a day (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e); FCR decreased from 1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 when two doses were fed and at 80% to 1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 when fed four doses (Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). However, this pattern was not the same at 100% of the ration, where FCR (1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13) was not significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) with two times a day from that obtained with four times a day (1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean (\u0026plusmn;\u0026thinsp;SD) feed conversion rate combined by frequencies (2 and 4 feedings) and ration sizes (80 and 100%) after 53 d of rearing juvenile Litopenaeus vannamei fed two different diets at two feeding frequencies and two satiation levels.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFrequency mean\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCombined\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 feeds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003ea B\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 feeds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u003csup\u003eb B\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRation mean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues for each diet-frequency-ration combination are means (\u0026plusmn;\u0026thinsp;standard deviation (SD) of three replicates.\u003c/p\u003e \u003cp\u003eThe frequency averages with different superscripts (uppercase per row and lowercase per column) are significantly different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eConsidering the average for two frequencies, reduction in ration size from 100 to 80% led to a lower combined FCR (1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 vs 1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07). The same pattern was also observed where FCR at 100% (1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13) was higher at two times per day than at 80% satiation (1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08) at four times per day when the ration decreased from 100 (1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11) to 80% satiation (1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07), which led also to a significant difference in FCR (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Attractant type effects\u003c/h2\u003e \u003cp\u003eNo statistical differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were observed in growth, feed conversion and survival between shrimp fed with the different marine hydrolysates. Nevertheless, the consumed FHIE was significantly higher than FHEE diet (107.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76 vs 99.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76, respectively), mainly in the two-meal frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditionally, regarding consumed feed, significant differences were observed (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between FHIE at 2-dose treatment and 100% of ration size, compared to all other treatments at 80% satiety and even compared to FHEE at 4-dose treatment and 100% ration size. The only exception was registered when FHIE at 2 doses and 100% was compared with FHIE at 2 doses and 80% satiety, where no significant differences were observed (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0,05\u003c/em\u003e), even though this treatment had a restriction of 20%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe present study found significant feeding frequency effects and ration restriction size on juvenile \u003cem\u003eL. vannamei\u003c/em\u003e growth and FCR. Feeding shrimp four times per day relative to twice a day significantly improved FCR but did not enhance growth; rather, a significantly lower growth was observed under this study conditions with four doses, that included night feeding, because feed consumption was reduced. These findings reveal the notable impact of feeding and ration size schedules on production efficiency when rearing shrimp, highlighting the need to understand this correlation when planning feed management schemes, as stated by Arnold et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Feeding frequency\u003c/h2\u003e \u003cp\u003eUnder the present study conditions, our results clearly show that increasing feeding frequency during night hours does not improve growth performance. Mean weight gain with two feedings per day (6.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 g) was significantly higher compared to four feedings per day (5.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 g), both at 100% and 80% ration sizes, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The highest results in weight gain were obtained when feed was administered during daylight hours (10:00 h and 16:00 h), while no additional benefit was observed when part of the ration was distributed during night hours (22:00 h and 04:00 h).\u003c/p\u003e \u003cp\u003eFindings indicate that \u003cem\u003eL. vannamei\u003c/em\u003e, as observed in studies by Nunes, Goddard \u0026amp; Gesterira (1996) and Reymond and Lagard\u0026eacute;re (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), exhibit different feeding behaviors compared to other penaeid shrimps. While most grooved penaeids are typically active and burrow during nighttime (Hindley, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1975\u003c/span\u003e), \u003cem\u003eL. vannamei\u003c/em\u003e showed to be more active in feeding during daylight hours as reported by Pontes et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and Nunes et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In terms of FCR more feedings per day resulted in more efficient feed utilization by shrimp as informed by Aalimahmoudi et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which is verified in lower feed conversion values.\u003c/p\u003e \u003cp\u003eGrowth improvement with two feed doses instead of four could be understood based on the enzyme profile activities throughout the day, suggesting an oscillatory pattern with feeding peaks at certain times when a maximum feeding activity exists.\u003c/p\u003e \u003cp\u003eIn crustaceans, certain biological phenomena have been observed to occur rhythmically around the same time (Casillas-Hern\u0026aacute;ndez et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Circadian rhythms affect enzymatic activity, which depends on several exogenous factors, such as age, size, protein sources, and molting and endogenous causes (such as ontogenics, metabolic rates, circadian rhythms) (Lemos, Ezquerra \u0026amp; Garc\u0026iacute;a-Carre\u0026ntilde;o 2000; Molina, Cadena \u0026amp; Orellana \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The results of the present study suggests that circadian rhythms might underlie variations in performance at different frequencies.\u003c/p\u003e \u003cp\u003eCasillas-Hern\u0026aacute;ndez et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) studied juvenile \u003cem\u003eL. vannamei\u003c/em\u003e enzymatic profile under continuous feed intake and found fluctuation in proteolytic activities. In an experiment with juvenile \u003cem\u003eL. vannamei\u003c/em\u003e, a biphasic circadian rhythm was observed with diurnal and nocturnal enzymatic peak activities at 10:00 and 20:00 h. Moreover, as observed, feeding two hours before these peaks (08:00 and 18:00 h) resulted in a significantly higher growth rate, final size, survival, and biomass. These findings suggest that some periods throughout the day exist in which feeding effort should be concentrated, and specific hours depend on \u003cem\u003eL. vannamei\u003c/em\u003e physiological status. The results also confirm synchrony between feeding activity and feed use.\u003c/p\u003e \u003cp\u003eThe biphasic circadian rhythm might explain why no effect was observed in growth when feeding frequency is increased at night. In the present study, shrimp fed two times per day had higher weight gain than those fed four times per day at 100% of ration size (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), which is also in agreement with Pontes, De Lima and Arruda (2008),Haz clic o pulse aqu\u0026iacute; para escribir texto. who reported that increased temporal space between feeding stimulates searching for and ingesting feed.\u003c/p\u003e \u003cp\u003eMoreover, regarding feeding schedule, Pontes and Arruda(2005) studied \u003cem\u003eL. vannamei\u003c/em\u003e feeding behavior as a function of artificial light and dark photoperiods and observed feeding time was higher in the light phase while swimming occurred mostly during the dark phase. In the same line, Sick, White and Baptist(1973) reported that ingestion rate was proportional to light intensity and inversely related to the time at which the feed was immersed in water in juvenile \u003cem\u003eLitopenaeus setiferus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, Robertson, Lawrence \u0026amp; Castille. (1993) reported that day feedings produce more growth in juvenile \u003cem\u003eL. vannamei\u003c/em\u003e held in pond enclosures than those at night feedings. Seemingly, \u003cem\u003eL. vannamei\u003c/em\u003e are inherently more active during day than at night.\u003c/p\u003e \u003cp\u003eMore recently, Reis et al. (2021) reported that in outdoor and indoor systems, findings suggest that \u003cem\u003eL. vannamei\u003c/em\u003e does not appear to have a specific feeding schedule preference as long as environmental conditions and overall feeding rates are appropriate. However, it is suggested that night feeding may be less efficient compared to day feeding, mainly due to limitations related to dissolved oxygen. During the night, the biological oxygen demand increases due to feeding activity, while oxygen levels are naturally lower. This leads to a higher risk of oxygen depletion, which may require adjustments such as reduced feed rations or more frequent use of mechanical aerators, which increases electrical costs.\u003c/p\u003e \u003cp\u003eIn terms of growth, shrimp fed exclusively at night or on a 24-hour schedule showed slightly slower growth and lower average final weights compared to day feeders or under the AQ1 (acoustic, on-demand) feeding system.\u003c/p\u003e \u003cp\u003eIn brief, an oscillating pattern in feeding habits suggests that an increase in feeding frequency should be based on natural behavior and physiological status with higher activity during daytime hours. Our findings show that growth can be sustained by reducing the feeding frequency if an excess amount of feed is provided to meet the nutritional requirements. However, it is important to note that this approach comes with a trade-off: feeding excess -even marginal- compromises feed efficiency (Arnold et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn terms of FCR, feeding shrimp four times daily resulted in a more efficient feed utilization (1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18) compared to feeding twice daily (1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16), as indicated in Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. These findings align with Aalimahmoudi et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), who also observed improved FCR with increased feeding frequencies. However, this improved efficiency did not translate into higher growth under our experimental conditions, suggesting that the benefits of increased feeding frequencies are context-dependent and should be evaluated in combination with environmental and operational factors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Ration Size\u003c/h2\u003e \u003cp\u003eThe present study demonstrates a detrimental effect of a restricted ration (20% below satiation) in juvenile \u003cem\u003eL. vannamei\u003c/em\u003e, even when feeding frequency increased from two to four times per day, because feed consumption was reduced. Feeding at apparent satiety -as demonstrated in the present study- led to an improved growth, since shrimp can consume feed allowing them to meet their nutritional requirements. Additionally, on demand feeding allows reducing stress associated with limited feeding. In this strategy, shrimp are less likely to compete for feed or exhibit aggressive behavior. Reduced stress levels can contribute to overall better health and disease resistance.\u003c/p\u003e \u003cp\u003eOn the other hand, ration size reduction to 80% improves feed efficiency, which was consistent for both diets, restrictive feeding offers optimal utilization of feed, although it comes at the expense of overall weight gain. Increased feed conversion ratios observed in the present study, such as growth rate increase, indicates that the feeding input plateau was likely approached (Weldon et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen the maximum weight gain is reached, an inflection point occurs at 101% of the feeding rate; beyond this point, growth and feed utilization decrease rapidly, resulting in a higher feed conversion ratio and diminished growth gains. Therefore, aiming for maximum gain is counteracted by the elevated FCR (Weldon et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur results are consistent with those of Arnold et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), who reported similar trends in FCR and growth when ration size was restricted. In this study Arnold et al.(2016) reported FCR decrease when ration reduced from 100% satiation (1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05) to 80% satiation (1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04). In agreement with our results, the present study also found a FCR reduction from 1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 at 100% satiation to 1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 at 80% ration size.\u003c/p\u003e \u003cp\u003eThe effect of reducing FCR as ration size decreases contrasts with that reported by Venero, Davis \u0026amp; Rouse(2007) where feeding at 100% and 75% led to significantly different yields for the two satiation levels: 6482 kg/ha at 100% versus 5054 kg/ha at 75%, respectively, but similar to FCRs for both treatments. On the other hand, the results obtained in the present study are very similar to those reported by Nunes et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), observing that feeding shrimp 4.5% of biomass with 0% restriction against 3.4% of biomass with 25% restriction resulted in improved growth performance with higher but still acceptable feed conversion rates. Nunes et al.(2006) found no differences between apparent satiation versus 25% restriction, which suggests the possibility of moderately reduce daily feeding without detrimental effects on growth. Nevertheless, an adverse effect was observed on growth performance at 20% feed restriction.\u003c/p\u003e \u003cp\u003eThe findings in the present study suggest that a restricted feeding scheme led to a reduced growth rate by limiting the feed available amount to shrimp. With this scheme, shrimp growth may be compromised. A restrictive feed scheme might create a competitive feeding environment, causing increased stress levels, aggression, and dominance behavior among shrimp. Stress can negatively affect their overall health and well-being. Moreover, restrictive feeding might lead to variations in individual growth rates within a population. Some shrimp may consume more feed than others because of dominant behaviors, leading to size disparities and uneven growth distribution within the group.\u003c/p\u003e \u003cp\u003eRestrictive feeding schemes also might not provide all the essential nutrients required for optimal shrimp growth and health which can impact various physiological functions. Efficient feeding practices ensure the right feed amount is provided. However, overfeeding should be avoided, thus, the release of excessive nutrient input in the water is important. By providing the precise feed quantity, farmers are able to minimize nutrients and organic matter release, reducing shrimp farming environmental impact (Boyd et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Weldon et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Type of diet\u003c/h2\u003e \u003cp\u003eNo significant differences were observed using either fish processed with internal (FHIE) or external enzymes (FHEE) in terms of growth and survival, but hydrolysates may enhance aquafeed palatability even though a variety of studies suggest that peptides with lower molecular weights facilitate easier absorption (Carvalho, S\u0026aacute;, Oliva-Teles \u0026amp; Bergot \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), since the hydrolysis process releases smaller peptides that can stimulate feeding response and improve feed intake.\u003c/p\u003e \u003cp\u003eFeed consumption was consistently higher in shrimp fed the FHIE diet, particularly at 100% ration size and two feedings per day (130.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47 g for FHIE vs. 116.77\u0026thinsp;\u0026plusmn;\u0026thinsp;4.11 g for FHEE). This suggests that the FHIE hydrolysate may enhance feed palatability, even though this did not translate into significant differences in growth or FCR under the present study conditions.\u003c/p\u003e \u003cp\u003eHydrolysates with a suitable peptide distribution in the 500\u0026ndash;1000 Da range appear to be effective in increasing feed palatability. Studies have shown that smaller peptides improve absorption efficiency and stimulate feeding responses. For example, Carvalho et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) found that low-molecular-weight peptides enhance nutrient utilization in fish larvae. Similarly, Kristinsson and Rasco (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) reported that hydrolysates with targeted peptide sizes improve palatability and functional properties. Aksnes et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) also demonstrated the role of size-fractionated fish hydrolysates in enhancing feed intake and efficiency in aquaculture diets, and finally, Hou et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) further highlighted that peptides within this molecular range are bioactive and functional, which may contribute to their effectiveness as feed attractants.\u003c/p\u003e \u003cp\u003eHydrolysates with a suitable peptide distribution in 500\u0026ndash;1000 Da range appear to be effectively increasing feed palatability, even though no effect in growth or survival is observed when FHIE is compared with FHEE. Feed consumption seems to be different for diets with two hydrolysates with an average of 99.5 g for FHEE and 107 g for FHIE, which is manifested in homologous diets, 117 vs 130 (2/100%), 95 vs 105 (2/80%), and 102 vs 106 (4/100%), except for 4/80%, which does not have an effect into growth or feed conversion. Further research is required to evaluate different peptide distributions in hydrolysates, as well as a narrower range of ration levels (100 and 90%) in combination of wider feeding frequency series (2, 4, 8, 16 times a day) followed by confirmation of the findings in commercial scenarios.\u003c/p\u003e \u003cp\u003eOptimal feeding strategies are essential to minimize feed wastage while ensuring adequate nutrient intake. Studies have shown that improper feed management can lead to increased production costs and environmental impact due to uneaten feed accumulation.\u003c/p\u003e \u003cp\u003eWhile hydrolysates can represent an additional cost in formulation, their ability to enhance feed intake and nutrient absorption may result in better overall profitability by reducing the total amount of feed required per unit of shrimp biomass produced (Hlordzi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSummarizing, the three aspects studied: type of feed, feeding frequency and ration size have significant implications on the biological performance of \u003cem\u003eL. vannamei\u003c/em\u003e and the final feed cost. In our study, it was observed that feeding twice a day resulted in higher growth, while four daily rations improved feed efficiency (FCR). From an economic perspective, these differences can translate into variations in management decisions and operational costs, such as feed use and labor in farms. Existing literature suggests that proper management of these variables can reduce costs associated with feed waste and energy used in nocturnal feeding, especially when consumption is lower (Reis et al. 2021). Thus, our recommendations highlight the importance of evaluating the relationship between operational cost and biological performance to design more profitable and sustainable feeding strategies.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe present study demonstrated that feed efficiency benefits of restricted ration are achieved but with less growth, even if feeding frequency was increased. The main outcomes highlight daylight feeding schedule significance and indicate that \u003cem\u003eL. vannamei\u003c/em\u003e shows no beneficial effect of restricting or increasing feeding frequency at night hours, and the positive effects of daylight feeding at hours close to juvenile \u003cem\u003eL. vannamei\u003c/em\u003e maximum enzymatic reported activities. The findings in the present research suggest that under laboratory conditions, feeding at higher level provides the most advantageous option for optimizing production performance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eCompeting interests:\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: MEO, RCC and MJC. Methodology: MEO, RCC and MJC. Formal analysis: MEO, CMP, RCC, MJC. Investigation: MEO, RCC. Writing - original draft preparation: MEO, RCC and MJC. Writing - review and editing: MJC, RCC, and CMP. Supervision: CMP, RCC, MJC.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful to the Ecuadorian Secretary of Higher Education, Science, Technology and Innovation support for providing the scholarship for Manuel Espinoza (Contract 063-2012). The authors also express their gratitude to Gisis S.A., as well as Sa\u0026uacute;l Zamora and Sandra de La Paz Reyes from the Aquatic Nutrition laboratory at CIBNOR, Mexico for the technical support during the feeding trial, and Diana Dorantes for English language edition.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAalimahmoudi, M., Reyshahri, A., \u0026amp; Bavarsad, S. S. (2016). Effects of feeding frequency on growth, feed conversion ratio, survival rate and water quality of white leg shrimp. \u003cem\u003eInternational Journal of Fisheries and Aquatic Studies\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(August 2015), 293\u0026ndash;297. https://www.fisheriesjournal.com/archives/2016/vol4issue3/PartD/4-2-88.pdf\u003c/li\u003e\n\u003cli\u003eAksnes, A., Hope, B., J\u0026ouml;nsson, E., Bj\u0026ouml;rnsson, B. T., \u0026amp; Albrektsen, S. (2006). Size-fractionated fish hydrolysate as feed ingredient for rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e) fed high plant protein diets. I: Growth, growth regulation and feed utilization. \u003cem\u003eAquaculture\u003c/em\u003e, \u003cem\u003e261\u003c/em\u003e(1), 305\u0026ndash;317. https://doi.org/10.1016/j.aquaculture.2006.07.025\u003c/li\u003e\n\u003cli\u003eArnold, S., Smullen, R., Briggs, M., West, M., \u0026amp; Glencross, B. (2016). The combined effect of feed frequency and ration size of diets with and without microbial biomass on the growth and feed conversion of juvenile \u003cem\u003ePenaeus monodon\u003c/em\u003e. \u003cem\u003eAquaculture Nutrition\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e(6), 1340\u0026ndash;1347. https://doi.org/10.1111/anu.12338\u003c/li\u003e\n\u003cli\u003eBerge, G. M., \u0026amp; Storebakken, T. (1996). Fish protein hydrolyzate in starter diets for Atlantic fry. \u003cem\u003eAquaculture\u003c/em\u003e, \u003cem\u003e145\u003c/em\u003e(00448486), 205\u0026ndash;212. https://doi.org/10.1016/S0044-8486(96)01355-5\u003c/li\u003e\n\u003cli\u003eB\u0026oslash;gwald, I., Herrig, S., Pedersen, A. M., Wubshet, S. G., \u0026amp; Eilertsen, K. E. (2024). Effect of Calanus finmarchicus Hydrolysate Inclusion on Diet Attractiveness for Whiteleg Shrimp (Litopenaeus vannamei). Fishes, 9(4). https://doi.org/10.3390/fishes9040134\u003c/li\u003e\n\u003cli\u003eBoyd, C. (2016). Consideraciones sobre la calidad del agua y del suelo en cultivos de camar\u0026oacute;n. M\u0026eacute;todos para mejorar la camaronicultura en Centroam\u0026eacute;rica p\u0026aacute;gs. 1\u0026ndash;30. \u003c/li\u003e\n\u003cli\u003eBoyd, C., Racine, P., McNevin, A., Ngo Minh, H., Quoc Tinh, H., Paungkaew, D., Viriyatum, R., \u0026amp; Engle, C. (2017). Resource Use Assessment of Shrimp \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e and \u003cem\u003ePenaeus monodon\u003c/em\u003e , Production in Thailand and Vietnam. \u003cem\u003eWorld Aquaculture Society\u003c/em\u003e, \u003cem\u003e48\u003c/em\u003e(2), 201\u0026ndash;226. https://doi.org/10.1111/jwas.12394\u003c/li\u003e\n\u003cli\u003eCarr, W. (1988). The Molecular Nature of Chemical Stimuli in the Aquatic Environment. \u003cem\u003eSensory Biology of Aquatic Animals\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e(1). https://doi.org/https://doi.org/10.1007/978-1-4612-3714-3_1\u003c/li\u003e\n\u003cli\u003eCarvalho, A. P., S\u0026aacute;, R., Oliva-Teles, A., \u0026amp; Bergot, P. (2004). Solubility and peptide profile affect the utilization of dietary protein by common carp (Cyprinus carpio) during early larval stages. \u003cem\u003eAquaculture\u003c/em\u003e. https://doi.org/10.1016/j.aquaculture.2004.01.007\u003c/li\u003e\n\u003cli\u003eCarvalho, E. A., \u0026amp; Nunes, A. J. P. (2006). Effects of feeding frequency on feed leaching loss and grow-out patterns of the white shrimp \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e fed under a diurnal feeding regime in pond enclosures. \u003cem\u003eAquaculture\u003c/em\u003e, \u003cem\u003e252\u003c/em\u003e(2\u0026ndash;4), 494\u0026ndash;502. https://doi.org/10.1016/j.aquaculture.2005.07.013\u003c/li\u003e\n\u003cli\u003eCasillas-Hern\u0026aacute;ndez, R., Nolasco-Soria, H., Lares-Villa, F., Garc\u0026iacute;a-Galano, T., Carrillo-Farnes, O., \u0026amp; Vega-Villasante, F. (2006). Ritmo circadiano de la actividad enzim\u0026aacute;tica digestiva del camar\u0026oacute;n blanco \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e y su efecto en el horario de alimentaci\u0026oacute;n. \u003cem\u003eRevista Latinoamericana de Recursos Naturales\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e(2), 55\u0026ndash;64. https://biblat.unam.mx/hevila/Revistalatinoamericanaderecursosnaturales/2006/vol2/no2/2.pdf\u003c/li\u003e\n\u003cli\u003eCivera, R., \u0026amp; Guillaume, J. (1989). Effect of sodium phytate on growth and tissue mineralization of \u003cem\u003ePenaeus japonicus \u003c/em\u003eand \u003cem\u003ePenaeus vannamei\u003c/em\u003e juveniles. \u003cem\u003eAquaculture\u003c/em\u003e, \u003cem\u003e77\u003c/em\u003e(2\u0026ndash;3), 145\u0026ndash;156. https://doi.org/10.1016/0044-8486(89)90198-1\u003c/li\u003e\n\u003cli\u003eC\u0026aacute;mara Nacional de Acuacultura CNA (22 de diciembre de 2023). https://www.cna-ecuador.com/estadisticas/\u003c/li\u003e\n\u003cli\u003eC\u0026oacute;rdova-Murueta, J. H., \u0026amp; Garc\u0026iacute;a-Carre\u0026ntilde;o, F. L. (2002). Nutritive value of squid and hydrolyzed protein supplement in shrimp feed. \u003cem\u003eAquaculture\u003c/em\u003e, \u003cem\u003e210\u003c/em\u003e(1\u0026ndash;4), 371\u0026ndash;384. https://doi.org/10.1016/S0044-8486(02)00011-X\u003c/li\u003e\n\u003cli\u003eFAO. 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Protein digestion in penaeid shrimp: Digestive proteinases, proteinase inhibitors and feed digestibility. \u003cem\u003eAquaculture\u003c/em\u003e, \u003cem\u003e186\u003c/em\u003e(1\u0026ndash;2), 89\u0026ndash;105. https://doi.org/10.1016/S0044-8486(99)00371-3\u003c/li\u003e\n\u003cli\u003eMolina, C., Cadena, E., \u0026amp; Orellana, F. (2000). Alimentaci\u0026oacute;n de camarones en relaci\u0026oacute;n a la actividad enzim\u0026aacute;tica como una respuesta natural al ritmo circadiano y ciclo de muda. In: Cruz-Su\u0026aacute;rez, L.E., Ricque-Marie, D., Tapia-Salazar, M., Olvera-Novoa, M.A. y Civera-Cerecedo. R, (Eds.). Avances en Nutrici\u0026oacute;n Acu\u0026iacute;cola V. Memorias del V Simposium Internacional de Nutrici\u0026oacute;n Acu\u0026iacute;cola. 19-22 Noviembre, 2000. M\u0026eacute;rida, Yucat\u0026aacute;n, M\u0026eacute;xico. P\u0026aacute;gs. 358-380\u003c/li\u003e\n\u003cli\u003eNapaumpaiporn, T., Chuchird, N., \u0026amp; Taparhudee, W. (2013). 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Feeding activity patterns of the southern brown shrimp \u003cem\u003ePenaeus subtilis\u003c/em\u003e under semi-intensive culture in NE Brazil. \u003cem\u003eAquaculture\u003c/em\u003e, \u003cem\u003e144\u003c/em\u003e(4), 371\u0026ndash;386. https://doi.org/10.1016/0044-8486(96)01297-5\u003c/li\u003e\n\u003cli\u003eNunes, A., S\u0026aacute;, M., Carvalho, E., \u0026amp; Sabry Neto, H. (2006). Growth performance of the white shrimp \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e reared under time and rate restriction feeding regimes in a controlled culture system. \u003cem\u003eAquaculture\u003c/em\u003e, \u003cem\u003e253\u003c/em\u003e(1\u0026ndash;4), 646\u0026ndash;652. https://doi.org/10.1016/j.aquaculture.2005.09.023\u003c/li\u003e\n\u003cli\u003eNunes, A., Sabry-Neto, H., Pires da Silva, F. H., Rodrigues de Oliveira-Neto, A. \u0026amp; Masagounder, K. (2019). Multiple feedings enhance the growth performance and feed efficiency of juvenile \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e when fed a low-fish meal amino acid-supplemented diet. \u003cem\u003eAquaculture International\u003c/em\u003e, \u003cem\u003e27\u003c/em\u003e(2), 337\u0026ndash;347. https://doi.org/10.1007/s10499-018-0330-7\u003c/li\u003e\n\u003cli\u003eNunes, C. R., Da Silva, C., Clovis, P., \u0026amp; Dos Santos, J. (2018). Evaluation of feeding rates in the production of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e shrimp using artificial substrates. \u003cem\u003eCiencia Animal Brasileira\u003c/em\u003e. https://doi.org/https://doi.org/10.1590/1809-6891v19e-50805\u003c/li\u003e\n\u003cli\u003ePontes, C. S., \u0026amp; Arruda, M. D. F. (2005). Comportamento de \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e (Boone) (Crustacea, Decapoda, Penaeidae) em fun\u0026ccedil;\u0026atilde;o da oferta do alimento artificial nas fases clara e escura do per\u0026iacute;odo de 24 horas artificial fases clara escura horas. \u003cem\u003eRevista Brasileira de Zoologia\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e(3), 648\u0026ndash;652.\u003c/li\u003e\n\u003cli\u003ePontes, C. S., Arruda, M. D. F., De Lara Menezes, A. A., \u0026amp; De Lima, P. P. (2006). Daily activity pattern of the marine shrimp \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e (Boone 1931) juveniles under laboratory conditions. \u003cem\u003eAquaculture Research\u003c/em\u003e, \u003cem\u003e37\u003c/em\u003e(10), 1001\u0026ndash;1006. https://doi.org/10.1111/j.1365-2109.2006.01519.x\u003c/li\u003e\n\u003cli\u003ePontes, C. S., De Lima, P. P., \u0026amp; Arruda M de F. (2008). Feeding responses of juvenile shrimp \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e (Boone) fed at different frequencies under laboratory conditions. \u003cem\u003eAquaculture Research\u003c/em\u003e, 1416\u0026ndash;1422. https://doi.org/10.1111/j.1365-2109.2008.02011.xKotusReis, J., Weldon, A., Ito, P., Stites, W., Rhodes, M., \u0026amp; Davis, D. A. (2021). Automated feeding systems for shrimp: Effects of feeding schedules and passive feedback feeding systems. Aquaculture, 541. https://doi.org/10.1016/j.aquaculture.2021.736800\u003c/li\u003e\n\u003cli\u003eReymond, H., \u0026amp; Lagard\u0026eacute;re, J. P. (1990). Feeding Rhythms and Food of \u003cem\u003ePenaeus japonicus\u003c/em\u003e Bate (Crustacea, Penaeidae) in Salt Marsh Ponds: Role of Halophilic Entomofauna. \u003cem\u003eAquaculture, 84 (1990) 125-143\u003c/em\u003e, \u003cem\u003e84\u003c/em\u003e, 125\u0026ndash;143.\u003c/li\u003e\n\u003cli\u003eRobertson, L., Lawrence, A. L., \u0026amp; Castille, F. (1993). Effect of feeding frequency and feeding time on growth of \u003cem\u003ePenaeus vannamei\u003c/em\u003e (Boone). \u003cem\u003eAquaculture and Fisheries Management 1993, 24, 1-6.\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eSick, L. V., White, D., \u0026amp; Baptist, G. (1973). The effect of duration of feeding, amount of food, light intensity, and animal size on rate of ingestion of pelleted food by juvenile penaeid shrimp. \u003cem\u003eProgressive Fish-Culturist\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e(1), 22\u0026ndash;26. https://doi.org/10.1577/1548-8659(1973)35[22:TEODOF]2.0.CO;2\u003c/li\u003e\n\u003cli\u003eSmith, D. M., Burford, M. A., \u0026amp; Tabrett, S. J. (2002). The effect of feeding frequency on water quality and growth of the black tiger shrimp (\u003cem\u003ePenaeus monodon\u003c/em\u003e). \u003cem\u003eAquaculture\u003c/em\u003e, \u003cem\u003e207\u003c/em\u003e, 125\u0026ndash;136.\u003c/li\u003e\n\u003cli\u003eTacon, A. G. J., Jory, D., \u0026amp; Nunes, A. (2013). Shrimp feed management: issues and perspectives. \u003cem\u003eFAO Fisheries and Aquaculture Technical\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e, 481\u0026ndash;488.\u003c/li\u003e\n\u003cli\u003eTan, B., Mai, K., Zheng, S., Zhou, Q., Liu, L., \u0026amp; Yu, Y. (2005). Replacement of fish meal by meat and bone meal in practical diets for the white shrimp \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e (Boone). \u003cem\u003eAquaculture Research\u003c/em\u003e, \u003cem\u003e36\u003c/em\u003e(5), 439\u0026ndash;444. https://doi.org/10.1111/j.1365-2109.2005.01223.x\u003c/li\u003e\n\u003cli\u003eVelasco, M., Lawrence, A., \u0026amp; Castille, F. (1999). Effect of variations in daily feeding frequency and ration size on growth of shrimp, \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e (Boone), in zero-water exchange culture tanks. \u003cem\u003eAquaculture\u003c/em\u003e, 179(1-4), 141-148\u003c/li\u003e\n\u003cli\u003eVenero, J. A., Davis, D. A., \u0026amp; Rouse, D. B. (2007). Variable feed allowance with constant protein input for the pacific white shrimp \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e reared under semi-intensive conditions in tanks and ponds. \u003cem\u003eAquaculture\u003c/em\u003e, \u003cem\u003e269\u003c/em\u003e(1\u0026ndash;4), 490\u0026ndash;503. https://doi.org/10.1016/j.aquaculture.2007.02.055\u003c/li\u003e\n\u003cli\u003eWeldon, A., Davis, D. A., Rhodes, M., Reis, J., Stites, W., \u0026amp; Ito, P. (2021). Feed management of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e in a high density biofloc system. \u003cem\u003eAquaculture\u003c/em\u003e, \u003cem\u003e544\u003c/em\u003e(April), 737074. https://doi.org/10.1016/j.aquaculture.2021.737074\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Feeding frequency, feeding strategies, hydrolysates, Litopenaeus vannamei, feed restriction","lastPublishedDoi":"10.21203/rs.3.rs-6251899/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6251899/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOptimum feed ratio and frequency ensure maximum growth and efficient feed utilization in all feeding management strategies. Thus, the present study evaluated the effects of feeding frequency and ration restriction on juveniles of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e (0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 g) fed two diets over 53 days. Feeding frequency included two (10:00, 16:00) and four times a day (10:00 h, 16:00, 22:00, 04:00 h), using isonitrogenous diets (35% protein) formulated with fish hydrolysates produced via external (FHEE) or internal (FHIE) enzymes. Feed was supplied at 100% and 80% of apparent satiation.\u003c/p\u003e \u003cp\u003eAt the end of the experiment, survival was not different among treatments (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Shrimp fed twice showed a significantly higher weight gain than those fed four times (6.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42 vs. 5.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 g, respectively); feed conversion ratio (FCR) was also higher (1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 vs 1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18, respectively) but not significantly different (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The results demonstrated an improved feed efficiency at 80% compared with 100% satiation (FCR\u0026thinsp;=\u0026thinsp;1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 vs 1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, respectively), which was achieved at growth expense (5.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 vs 6.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51, respectively). No differences in weight gain were observed when comparing the distinct types of diets (FHEE or FHIE). The primary outcomes of the present study indicate a detrimental effect on reduced ration size growth at 80% without any benefits of increasing feeding frequency at night hours. The results of the present study also highlight the impact of apparent satiation and daylight feeding schedules in juvenile \u003cem\u003eL. vannamei\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Combined feeding frequency and ration size effects on juvenile Litopenaeus vannamei performance fed diets supplemented with fish hydrolysates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-28 15:37:17","doi":"10.21203/rs.3.rs-6251899/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7918c13e-d2c9-4d56-8b34-7d5d3b53c06b","owner":[],"postedDate":"March 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-06T08:38:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-28 15:37:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6251899","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6251899","identity":"rs-6251899","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}