Optimizing white shrimp aquaculture: Feeding frequency and automatic feeders impact on growth and antioxidant capacity

doi:10.21203/rs.3.rs-3219841/v1

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Optimizing white shrimp aquaculture: Feeding frequency and automatic feeders impact on growth and antioxidant capacity

https://doi.org/10.21203/rs.3.rs-3219841/v1

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The feeding regime is a critical aspect of white shrimp (Litopenaeus vannamei) aquaculture, but research on intensive shrimp culture using automatic feeders is scarce. Our study investigated different feeding strategies for adult white shrimp in intensive aquaculture tanks, evaluating growth, survivability, feed utilization, shrimp digestive and antioxidant capacity, and conducting an economic analysis. Shrimp (8.0 ± 0.5 g) were cultured for 35 days with feeding frequencies of 6, 8, 10, and 12 times/day (A6, A8, A10, and A12 groups, respectively) using automatic feeders, and one group was fed manually 6 times/day (M6). The results show that the automatic feeding groups had a higher final body weight and specific growth rate, and a lower feed conversion ratio compared to the manual group. Among the automatic feeding groups, A6 and A8 exhibited the best performance, with a quadratic regression suggesting 7.83 times/day as an optimal feeding frequency. Digestive enzyme activity (TP and α-AMS) was significantly higher in the A6 and A8 groups (p < 0.05). However, the A6 and A8 groups showed significantly higher levels of antioxidant enzymes (SOD and GPx) and lower levels of lipid oxidant enzyme (MDA), indicating better digestive and antioxidant abilities in shrimp with higher growth rates. Moreover, the antioxidant genes expression of MnSOD and GPx testified the enzyme changing. Pearson's correlation confirmed the positive correlation between SOD and GPx. Economically, the A8 group showed the highest profitability among all groups. Overall, the study recommends a feeding frequency of 6–8 times/day using automatic feeders as a suitable and feasible approach for white shrimp culture in intensive settings.

White shrimp

Aquaculture

Feeding frequency

Automatic feeders

Growth

Antioxidant capacity

Feeding plays a crucial role in intensive shrimp aquaculture and represents approximately 60% of the total production costs (Asche and Smith, 2018). The goal of optimal feed management is to prevent overfeeding, minimize waste, prevent cannibalism, and enhance production efficiency (Carvalho and Nunes, 2006). The feeding method employed should be tailored to meet the physiological and growth needs of intensively reared shrimp.

White shrimp, Litopenaeus vannamei (Boone), are widely consumed. In 2020, over 5400,000 tonnes of white shrimp were produced globally, making up approximately 80% of total crustacean production (FAO, 2022). Rapid growth (three months from post-larva to commercial adult), good survival in high-density conditions, and high disease tolerance make white shrimp a good choice for intensive and/or bio-secure closed grow-out systems.

Feeding frequency is crucial to ensuring a good feeding method (Salgado-Ismodes et al., 2020). An optimal feeding frequency helps improve shrimp growth and development, and enhance the feeding and feed utilization efficiency of shrimp. At the same time, it can reduce feed coefficient, heterogeneity, and environmental pollution, thereby improving total production and economic benefits. When shrimp are raised at a high density in industry shrimp culture, feeding management requires more effort as there is greater competition among L. vannamei that limits the yield of shrimp (Sanchez et al., 2005).

Automation of feeding is essential in high-density, large-scale shrimp culture to overcome the time and labor constraints associated with manual feeding. This study compares automated and manual feeding methods and examines the impact of feeding frequency using automatic feeders. The research contributes to the existing progress in optimizing feeding frequency for shrimp culturing, offering valuable insights for enhancing efficiency and sustainability in aquaculture practices.

Pěnka et al. (2023) found that 8-hour intervals were optimal for Sander lucioperca growth and welfare. Further, Biswas et al. (2023) reported that when fed three times/day the growth and survival rates and the economic return of Mystus gulio in brackish water net cages were optimal. Wang et al. (2022) found that feeding twice daily resulted in higher FBW and SGR values than fish fed one, three, or four meals daily. Meanwhile, Wu et al. (2021) discovered that 3 times/day was suitable for the growth and metabolism-related fgf21 expression of Ctenopharyngodon idellus. Despite the significance of intensive shrimp aquaculture, there remains a lack of sufficient data on the relevant aspects. This study addresses this gap and provides valuable insights for shrimp culturists to minimize feed loss and develop efficient feeding strategies. Overfeeding in intensive systems can lead to water quality degradation, decreased feed digestion efficiency, and increased waste production, highlighting the importance of utilizing this information to optimize feed usage and reduce expenses (Pedrosa et al., 2019). Automatic feeder systems (timer or computer-controlled) require such information for maximum efficiency.

Automatic feeders are extensively employed in intensive aquaculture, often setting feeding rations and frequencies to maximize fast growth, even at the cost of using excess feed. However, this study emphasizes the importance of focusing feeding strategies on physiological needs rather than solely on rapid growth performance in aquafarming practices (Pedrosa et al., 2019). Hence, it is important to further optimize the feeding frequency, taking into account the physiological adaptability and welfare of aquatic species.

This experiment conducted feeding studies under conditions simulating commercial production plants to ensure the relevance of the results to the shrimp aquaculture industry. Various feeding frequencies were tested, allowing evaluation of their impact on shrimp growth performance and health indicators, such as digestive enzyme activity, antioxidant levels, and related gene expressions. The study aims to provide scientific data for the application of automatic feeders to promote healthy shrimp cultivation in intensive aquaculture settings.

2.1 Experimental shrimp

The experimental shrimp were collected from Tongwei Aquatic Technology Co., Ltd. (Dongying, Shandong, China). When the body weight of L. vannamei reached approximately 8 g, randomly selected healthy shrimp were starved for 1 day and placed in a 1.5 m deep, 18 m² surface industry aquaculture tank for feeding, with a stocking density of 300 orgs/m³ and 1 m water depth (This stocking density helps prevent crowding stress and promotes normal growth (Liu et al., 2017)).

2.2 Experimental design and management

This study comprised six experimental groups, including one manual feeding control group (M6) and four automatic feeding groups with varying frequencies: 12 times/day (A12), 10 times/day (A10), 8 times/day (A8), and 6 times/day (A6). Each group consisted of three parallel tanks, resulting in a total of 15 tanks in the industry plant. The commercial automatic feeding machine used in the study was supplied by Huixintai Co., Ltd. (Dalian, China).

The daily feeding amount, water exchange rate, feeding method, and other conditions of each experimental group were consistent, and the feeding time was controlled within 20 min to finish. The experiment lasted 35 days. The seawater was purified using sand filtration and UV sterilization equipment. Water changes were conducted daily, and the water volume replaced ranged from 1/5 to 1/2 of the total volume, based on daily water quality assessments. During the experiment, the weighing of L. vannamei was conducted every 7 days. Feed amounts were recorded for the calculation of growth performance and yield.

2.3 Sampling

After 35 days of standardized cultivation, the L. vannamei were harvested, weighed, and the yield, WGR, SGR, and FCR were recorded. Twenty shrimps from each group were randomly harvested and euthanized using thermal shock for final sampling, which included recording the average body weight and tissues.

The hepatopancreatica were rinsed, dried, and collected in a 5 mL cryopreservation tube. Subsequently, the samples were frozen in liquid nitrogen and stored at a low temperature (-80℃) for the determination of digestive enzymes and antioxidant enzyme activities.

The Institutional Animal Care and Use Committee (IACUC) of Zhejiang University (Zhejiang, China) approved this study. All sampling procedures adhered to the guidelines of the IACUC on the care and use of animals for scientific purposes.

2.4 Growth performance

The growth performance was calculated using the recorded data according to the following equations:

WGR:

$$\text{W}\text{G}\text{R}=\frac{{W}_{2} -{W}_{1}}{{W}_{1}}$$

SGR:

$$\text{S}\text{G}\text{R}=\frac{{lnW}_{2} -{lnW}_{1}}{{t}_{2}-{t}_{1}}\times 100$$

FCR:

$$\text{F}\text{C}\text{R}=\frac{feed supply \left(kg\right)}{fish biomass increase \left(kg\right)}$$

Survival (SUR):

$$\text{S}\text{U}\text{R}=\frac{{N}_{2} -{N}_{1}}{{N}_{1}}$$

W₁ and W₂ represent the initial body weight (t₁) and final body weight (t₂) respectively. N₁ indicates the initial number of shrimps, while N₂ represents the final number of shrimps.

2.5 Determination of digestive enzyme and antioxidant enzymes activities in the hepatopancreas.

The hepatopancreatica were taken from a -80 ℃ refrigerator and thawed in a 4 ℃ refrigerator. The hepatopancreatica in each group was pooled and placed in a 10 mL centrifuge tube. Physiological saline was added in a certain proportion, and the samples were thoroughly crushed with a tissue crusher (HUXI, FJ200-SH, Shanghai, China). Then, the crushed samples were divided into 1.5 mL enzyme-free centrifuge tubes and operated on ice. They were subsequently centrifuged at 4 ℃ for 10 min at 4,500 g, and the supernatant was carefully collected and refrigerated for later use. The activity indicators of digestive enzymes, such as TP and amylase (AMS), including α and β-AMS in the intestine, were determined. Additionally, antioxidant enzyme activity indicators such as MDA, SOD, and GPx were tested following the kit instructions (Nanjing Jiancheng Biotechnology Research Institute).

2.6 Antioxidant genes expression

Quantification of gene expression was conducted using 15 samples per treatment, obtained by dividing the pools. RNA was quantified spectrophotometrically and evaluated for quality using agarose gel electrophoresis (1% w/v). Residual DNA was removed from 5 µg of total RNA using 1 U DNAse (Invitrogen) at 37°C for 60 min, followed by inactivation with 15mM EDTA at 75°C for 10 min. For relative expression analysis, 1.0 µg of RNA from experimental hepatopancreas was mixed with 0.5 µg oligo-dT, incubated at 70°C for 5 min, and then chilled on ice for 5 min. Retro transcription was performed using 1 µL of ImProm-II reverse transcriptase (Promega), 4 µL buffer 5 × ImProm-II, 30mM MgCl₂, 0.5mM dNTP, and 40 U RNAsin (Promega) in a 20 µL reaction, incubated at 25°C for 5 min, extended at 42°C for 60 min, and inactivated at 70°C for 15 min.

The expression of MnSOD and GPx genes was analyzed using a fluorescent quantitative detection system (Eppendorf Mastercycler ep realplex2 S). Each reaction consisted of 3 µL GoTaq Flexi Buffer, 2.5mM MgCl₂, 0.15mM dNTPs, 0.45 U GoTaq Flexi DNA polymerase (Promega), and 1× EvaGreen DNA-binding dye (Biotium; Hayward, CA, USA) in a final volume of 15 µL (Llera-Herrera et al., 2012). The primer pairs and their concentrations for each gene are provided in Table 1. Reverse PCR (RT-qPCR) analysis was performed with an initial step at 94°C for 3 min, followed by 40 cycles at 94°C for 10 s and 60°C for 30 s. A dissociation curve was generated in the range of 60°C to 94°C to confirm the specificity of the amplified products. All samples were analyzed in triplicate, and relative gene expression levels were calculated as previously described (Ji et al., 2009). The RT-qPCR efficiency of each primer pair (Table 1) was determined by constructing standard curves using 10-fold cDNA dilutions ranging from 1:10 to 1:100,000. The efficiency of the PCR reaction (E) was calculated from the slope of the standard curve using the formula E = 10 (− 1/slope) -1. The Elongation Factor (EF1A) gene was used as an internal control to normalize the expression levels of the target genes.

Table 1

Primer pairs and the concentration for each qPCR testing
Gene	Primer	Concentration (µM)	Efficency (%)
EF1A	5′-ATGGTTGTCAACTTTGCCCC-3′	120	92.13	Sittikankaew et al. (2010)
	5′-TTGACCTCCTTGATCACACC-3’
MnSOD	5′-TCATGCTTTGCCACCTCTC-3′	90	95.62	Ji et al. (2009)
	5′-CCGCTTCAACCAACTTCTTC-3′
GPx	5′-GAAGACCCGGTGACCCAAAA-3′	80	91.43	Guzmán-Villanueva et al. (2020)
	5′-TTCTGTTTCTCCGCTCTCCG-3′			Guzmán-Villanueva et al. (2020)

2.6 Data analysis

Statistical analysis was conducted using IBM SPSS 20.0 for Windows (IBM Corporation, New York, NY, USA). Significance was determined at p < .05. One-way analysis of variance (ANOVA) was utilized to identify significant differences in growth parameters, digestive parameters, and antioxidant parameters. Tukey's test at the p < .05 level of significance was used for post-hoc analysis to determine group differences. The Pearson’s correlation among physiological enzymes, gene expression, and growth parameters was calculated and visualized using R 4.3.0. in the corrplot package 0.92 and pheatmap package.

3.1 Growth performance of L. vannamei and suitable feeding frequency

During the entire experiment, the water quality was controlled (Table 2). Shrimp growth was heightened in the automatic feeder group as there was a larger feeding area. Figure 1 exhibits the automatic feeding machine and its fan-shaped spraying. The scope of feed sprinkling was wider and covered most of the tank. Moreover, in the shrimp production plant, higher feeding frequencies (> 6 times/day) were commonly employed, and the evident advantage of labor-saving was observed with the use of automatic feeding machines.

Table 2

Mean (min, max) of normal water quality parameters of the five experimental treatments of *L. vannamei* during the 35-day experimental period.
	Treatments
	M6	A6	A8	A10	A12
T (℃)	29.33 ± 1.21	29.11 ± 1.12	29.45 ± 1.31	29.89 ± 1.29	29.76 ± 1.14
	(24.70, 32.86)	(25.57, 31.41)	(24.74, 32.63)	(26.31, 33.42)	(27.10, 32.74)
Salinity (g L^− 1)	27.21 ± 2.11	27.39 ± 3.20	27.19 ± 3.13	27.10 ± 2.97	27.43 ± 3.21
	(23.01, 29.10)	(23.09, 30.11)	(22.89, 30.32)	(23.09, 29.09)	(22.65, 29.87)
DO (mg L^− 1)	6.37 ± 0.39	6.29 ± 0.21	6.32 ± 0.43	6.56 ± 0.33	6.24 ± 0.41
	(6.10, 6.60)	(6.00, 6.50)	(5.90, 6.70)	(5.90, 6.60)	(5.80, 6.80)
pH	8.10 ± 0.15	8.16 ± 0.11	8.13 ± 0.09	8.15 ± 0.16	8.17 ± 0.20
	(8.00, 8,20)	(7.98, 8.32)	(7.90, 8.31)	(8.10, 8.30)	(7.94, 8.40)
NH₄-N (mg L^− 1)	3.24 ± 1.20	3.61 ± 1.14	3.57 ± 1.34	3.82 ± 1.05	3.86 ± 1.28
	(0.52, 4.67)	(0.63, 4.89)	(1.02, 5.20)	(1.22, 5.13)	(2.02, 6.03)
NO₂-N (mg L^− 1)	2.13 ± 0.85	2.3 ± 0.92	2.55 ± 1.64	2.65 ± 1.69	2.80 ± 1.71
	(0.32, 3.31)	(0.52, 4.10)	(0.59, 5.03)	(0.67, 6.60)	(0.74, 6.65)
NO₃-N (mg L^− 1)	10.22 ± 5.25	11.20 ± 3.12	20.32 ± 8.78	30.51 ± 9.12	35.61 ± 9.76
	(2.11, 15.23)	(2.23, 16.10)	(3.55, 25.63)	(4.12, 40.87)	(4.02, 42.21)
T, temperature; Sal, salinity; DO, dissolved oxygen; NH₄-N, ammonia; NO₂-N, nitrite; NO₃-N, nitrate

We observed that the A6 and A8 groups exhibited a lower FCR (Table 3), indicating more efficient feeding in these groups. However, when the feeding frequency was increased to 12 times/day, the FCR also increased, suggesting reduced feed utilization. This finding aligns with previous reports that optimal feeding frequency results in lower FCR and higher FBW in aquatic animals (Pěnka et al., 2023; Wu et al., 2021). Higher feeding frequency may lead to poor water quality, adversely affecting animal health and subsequently reducing growth performance, consistent with our study. Additionally, unsuitable feeding frequency was associated with abnormal metabolism (Wu et al., 2021).

Table 3

Growth performance of *L. vannamei* in the 35-day trial
	Treatments
	M6	A6	A8	A10	A12
IBW (g)	8.26 ± 2.43^a	8.22 ± 1.85^a	8.24 ± 2.35^a	8.29 ± 1.89^a	8.21 ± 2.10^a
FBW (g)	18.89 ± 2.93^a	19.88 ± 2.39^b	20.45 ± 2.56^b	20.32 ± 3.09^b	20.11 ± 2.33^b
SGR (%)	11.82 ± 1.25^a	12.62 ± 1.43^b	12.98 ± 1.13^b	12.81 ± 1.88^b	12.80 ± 1.17^b
FCR	1.43 ± 0.01^a	1.32 ± 0.05^b	1.30 ± 0.08^b	1.33 ± 0.02^b	1.40 ± 0.03^a
SUR (%)	96.40 ± 1.40^a	98.42 ± 0.31^b	98.48 ± 1.35^b	96.53 ± 1.33^a	95.4 ± 1.25^a
Yield (kg tank^− 1)	98.33 ± 5.63^a	105.66 ± 6.42^b	108.75 ± 7.32^b	105.92 ± 4.21^b	103.6 ± 5.21^b
Different superscript means significant difference (p < .05) (n = 3). IBW, initial body weight; FBW, final body weight; SGR, special growth rate; FCR, feed conversion ratio; SUR, survival.

Table 3 shows that FBW in all the automatic feeder groups was higher. Among the automatic feeding groups, FBW in the A8 and A10 groups was significantly higher than the other groups. FCR was highest in the M6 and A12 groups, followed by the A10 group. A6 and A8 did not exhibit any significant difference (p > .05). These results indicate that the optimal feeding frequency is between 6 and 8 times/day. According to the quadratic regression equation of FCR, the optimal feeding frequency is 7.83 times/day (Fig. 2), indicating that the A8 group may exhibit the most optimal results. Higher feeding frequency increases labor costs, but low feeding frequency will lead to decreased feed utilization and growth performance. The automatic feeder is both labor-saving and also has a wider distribution area.

After 21 days, the body weight in each week showed that the A8 group had the highest values among all treatments (Table 4) and was significantly higher than the M6 group (p < 0.05). The A6 group was also significantly higher than the M6 group, but only at the end of the study (p < .05).

Table 4

Body weight of *L. vannamei* at each stage of the weekly growth cycle.
Days	Treatments
Days	M6	A6	A8	A10	A12
1	8.26 ± 1.04^a	8.22 ± 1.25^a	8.24 ± 1.01^a	8.29 ± 1.00^a	8.21 ± 0.87^a
7	10.73 ± 1.12^a	10.88 ± 1.23^ab	11.28 ± 1.13^b	11.3 ± 1.08^b	11.21 ± 1.06^b
14	13.46 ± 1.59^a	13.56 ± 1.52^a	13.75 ± 1.19^a	13.7 ± 1.15^a	13.62 ± 1.12^a
21	15.22 ± 1.88^a	15.57 ± 1.78^a	15.9 ± 1.65^a	15.77 ± 1.29^a	15.67 ± 1.22^a
28	16.89 ± 2.10^a	17.66 ± 2.09^ab	18.02 ± 2.17^b	17.91 ± 2.20^b	17.76 ± 2.16^b
35	18.89 ± 2.93^a	19.88 ± 2.39^b	20.45 ± 2.56^b	20.32 ± 2.09^b	20.11 ± 2.01^b
Different superscript means significant difference (p < .05) (n = 3).

Early on in the experiment (7–14 days), the WGR was almost the same between the M6 and A6 groups and among the A8, A10, and A12 groups (Table 5). However, in the later stage of the experiment (after 21 days), A8 had the highest value among all the treatments. The highest WGR was observed in A8 at the end of the experiment (35 days). Interestingly, the WGR in the A12 group was almost identical to that of the A8 and A10 groups. However, after 28 days, the WGR in the A8 group gradually surpassed that of the other automatic feeding groups.

Table 5

Weight gain rate of *L. vannamei* at each stage of the weekly growth cycle.
Days	Treatments
Days	M6	A6	A8	A10	A12
7	29.90 ± 2.29^a	32.36 ± 2.10^a	36.89 ± 1.90^c	36.31 ± 2.18^c	36.54 ± 2.12^c
14	25.44 ± 2.12^a	24.63 ± 1.87^a	21.9 ± 1.28^b	21.24 ± 1.98^b	21.50 ± 2.22^b
21	13.08 ± 1.87^a	14.82 ± 1.35^ab	15.64 ± 1.96^b	15.11 ± 1.76^b	15.05 ± 1.08^b
28	10.97 ± 2.10^a	13.42 ± 1.88^b	13.33 ± 2.02^b	13.57 ± 1.67^b	13.30 ± 1.45^b
35	11.84 ± 1.21^a	12.57 ± 1.08^b	13.49 ± 2.07^bc	13.46 ± 1.12^c	13.23 ± 1.07^c
Different superscript means significant difference (p < .05) (n = 3).

At the end of the experiment, FBW was the highest in the A8 group (20.45 ± 2.56 g), although there was no significant difference among the automatic groups at the end (p > .05) (A6, A8, A10, and A12 groups). Meanwhile, A8 had the lowest FCR (1.30 ± 0.08), and the highest survival (98.48 ± 1.35%) and yield (108.75 ± 7.32 kg tank^− 1). These results indicate that during the initial stages of the experiment, the growth difference among shrimp in different feeding frequency treatments was not significant. However, after three weeks into the experiment, the difference became apparent, highlighting the crucial role of feeding frequency as an impactful factor in long-term shrimp production.

3.2 Effect of feeding frequency on the digestive enzymes in the hepatopancreas

L.vannamei, being a crustacean, differs from vertebrates as it combines the functions of the intestine, liver, and pancreas in the hepatopancreas. The activity of digestive enzymes in the hepatopancreas serves as a primary indicator of shrimp digestive function and growth metabolism (Vogt, 2019), reflecting their ability for nutrient absorption. In this study (Fig. 3a, b, and c), the activities of TP, α-AMS, and β-AMS in the hepatopancreas were measured at the end of the experiment. TP was significantly higher in the A6 group (p < 0.05), while α-AMS was significantly higher in the A8 group (p < 0.05). β-AMS activity varied significantly among the five treatments (p > .05). These results suggest that a suitable feeding regiment benefits digestive capability, which is consistent with the better growth performance observed in the A6 and A8 groups. Previous studies (Ettefaghdoost and Haghighi, 2021); Fawzy et al. (2022); (Wang et al., 2018) have reported similar findings, where increased digestive enzyme activity after specific feeding regimes correlated with enhanced growth performance in L. vannamei and other aquatic animals. Hence, in this study, feeding frequency emerged as the primary factor influencing digestive enzymes and growth in L. vannamei.

3.3 Effect of feeding frequency on the antioxidant enzymes and relative genes expression in the hepatopancreas

We observed that following 35 days of feeding in the A8 group, SOD and GPx activities were significantly increased in the hepatopancreata (Fig. 3d and e). Higher levels of antioxidant enzymes, such as SOD or GPx, typically indicate enhanced antioxidant capability. Previous studies investigating feeding supplementation, including probiotics or other additives, consistently observed higher SOD enzyme levels (Chien et al., 2023; Chiu et al., 2007; Fawzy et al., 2022). Researchers reported that in high-stress contexts, the organism copes through various functional adaptations for radical scavenging, which improves their health by increasing SOD levels. For example, in a low-salinity stress situation, L. vannamei will increase SOD levels. The researchers hypothesized that the higher the value, the more active the antioxidant capability in the group. This is consistent with the evidence that antioxidative enzymes prevent tissue or cell damage by increasing SOD and GPx values (Li et al., 2008; Mukherjee et al., 2022).

In the A8 group, significantly reduced MDA values were observed (p < 0.05) (Fig. 3f), which indicates lower lipid peroxidation. Muttharasi et al. (2021) observed lower MDA values in the higher antioxidant group fed with Amphiroa fragilissima crude polysaccharides encapsulated in Artemia nauplii. These results suggested that feeding 8 times/day could improve the antioxidant capacity and reduce the oxidative stress level of L. vannamei.

The production of antioxidant enzymes is regulated by antioxidant genes, and their relative expression levels serve as indicators of the corresponding antioxidant enzyme content. Previous studies, such as Liu et al. (2018), have demonstrated that higher levels of SOD were observed in healthy groups with the addition of commercial probiotics. In our study (Fig. 4a), significantly higher MnSOD gene expression was observed in the A6 and A8 groups (p < 0.05), which correlated with the higher SOD enzyme content. This finding confirms that higher gene expression is associated with increased enzyme activity. Additionally, after the 35-day experimental period, we observed significantly higher GPx transcript levels in the A6 group (p < 0.05) (Fig. 4b). The glutathione system plays a vital role in maintaining cellular redox status and detoxification. GPx facilitates the reduction of H₂O₂ to H₂O, working alongside SOD to remove free radicals (Vijayavel et al., 2004). Therefore, the higher expression levels of MnSOD and GPx transcripts in the A6 and A8 groups further support their enhanced antioxidant capability. These gene expression results are consistent with the observed changes in antioxidant enzyme levels, providing evidence for the dynamic changes in antioxidant enzymes.

Pearson’s correlation analysis is shown in Fig. 5, showing a positive relationship between SOD and MnSOD with GPx and GPx transcript levels, further supporting the cooperative role of SOD and GPx in antioxidant capacity, as previously discussed. Interestingly, the changing pattern of MDA was found to be negatively correlated with SOD and GPx, indicating a distinct mechanism for MDA compared to SOD and GPx. Additionally, FBW showed a positive correlation with SOD and GPx, indicating that improved antioxidant capability corresponded to more rapid shrimp growth performance, consistent with previous reports (Liu et al., 2018; Liu et al., 2017).

Overall, the digestive and antioxidant capacity of shrimp hepatopancreatica was improved in the A8 group, manifested as decreased MDA values and increased SOD, MnSOD, GPx, and GPx transcript.

3.4 Rough economic analysis

Based on the results (Table 6), it is evident that the highest feeding frequency and the manual feeding mode are not necessarily the most economical methods for rearing L. vannamei. A. The most cost-effective feeding frequency was observed in the A8 group. Moreover, manual feeding proved to be time-consuming, labor-intensive, and costly, often yielding unsatisfactory results. This study emphasizes that using an automatic feeder is a valuable tool in intensive shrimp aquaculture, with a feeding frequency of 8 times/day being identified as the optimum approach.

Table 6

Cost per kilogram of yield for *L. vannamei* cultured in the five treatments during the 35-day trial.
	Treatments
	M6	A6	A8	A10	A12
Manual work (yuan kg^− 1 shrimp)	3.39 ± 0.19^a	3.15 ± 0.21^ab	3.07 ± 0.31^b	3.15 ± 0.21^ab	3.22 ± 0.15^b
Energy for water exchange (yuan kg^− 1 shrimp)	3.49 ± 0.18^a	3.79 ± 0.12^ab	3.83 ± 0.11^ab	4.09 ± 0.18^b	4.34 ± 0.21^b
Energy for aeration (yuan kg^− 1 shrimp)	1.71 ± 0.03^a	1.59 ± 0.02^a	1.54 ± 0.05^a	1.59 ± 0.05^a	1.62 ± 0.03^a
Food consumption (yuan kg^− 1 shrimp)	8.80 ± 0.12^a	8.85 ± 0.11^a	8.91 ± 0.31^a	8.84 ± 0.12^a	8.88 ± 0.22^a
Probiotic consumption (yuan kg^− 1 shrimp)	2.03 ± 0.21^a	1.89 ± 0.31^a	1.84 ± 0.17^a	1.89 ± 0.24^a	1.93 ± 0.20^a
Total cost (yuan kg^− 1 shrimp)	14.81 ± 0.98^a	14.78 ± 0.32^a	14.74 ± 0.12^a	15.07 ± 0.10^ab	15.48 ± 0.18^b
Total cost (dollar kg^− 1 shrimp)	2.04 ± 0.02^a	2.05 ± 0.03^a	2.04 ± 0.01^a	2.09 ± 0.03^a	2.14 ± 0.04^a
Different superscript means significant difference (p < .05) (n = 3). (yuan = Chinese currency unit; US$1 = 7.1663 yuan)

In summary, maintaining an optimal feeding frequency has been shown to enhance the growth performance, antioxidant capacity, and cost-effectiveness of L. vannamei production. Utilizing multiple smaller meals is a widely employed approach in shrimp farming, and the implementation of automatic feeding systems can greatly improve the efficiency of shrimp aquaculture while reducing labor requirements.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author Contributions

Qinlang Liang and Gang Liu conceived, planned, designed, and performed the data analyses and the experiments. Qinlang Liang performed the bioinformatics analysis of q-PCR. Songming Zhu, Zhangying Ye, Xingguo Liu, Jian Zhao and Ying Liu conceptualized, designed, and supervised the study. Qinlang Liang and Gang Liu wrote the manuscript. Songming Zhu gave the revising suggestion of this paper. All authors contributed to the final paper.

Funding

This work was supported by the National Key R&D Program of China [Grant No. 2022YFD2001700], the Key Program of Science and Technology of Zhejiang Province [Grant No. 2023C02050]. The Key Laboratory of Environment Controlled Aquaculture, Ministry of Education [Grant No. 2021-MOEKLECA-KF-09]. Municipal and school cooperation project, Zhoushan Science and Technology Bureau [Grant No. 2022C81009].

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No competing interests reported.

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Optimizing white shrimp aquaculture: Feeding frequency and automatic feeders impact on growth and antioxidant capacity

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Experimental shrimp

2.2 Experimental design and management

2.3 Sampling

2.4 Growth performance

2.5 Determination of digestive enzyme and antioxidant enzymes activities in the hepatopancreas.

2.6 Antioxidant genes expression

2.6 Data analysis

3. Results and discussion

3.1 Growth performance of L. vannamei and suitable feeding frequency

3.2 Effect of feeding frequency on the digestive enzymes in the hepatopancreas

3.3 Effect of feeding frequency on the antioxidant enzymes and relative genes expression in the hepatopancreas

3.4 Rough economic analysis

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