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Effect of Two Microecological Preparations on the Breeding of White Leg Shrimp, Litopenaeus vannamei.

Byline: Song Jiang, Fa-Lin Zhou, Jian-Hua Huang, Qi-Bin Yang and Li-Shi Yang

Keywords: Litopenaeus vannamei, Microecological preparation, Breeding, Body weight, Survival rate, stress resistance.


Litopenaeus vannamei, also known as Penaeus vannamei, has experienced rapid development in the Chinese aquaculture industry since its introduction in the 1990s (Yao et al., 2007). To control the massive environmental pressure of high density cultures, many farmers have injected huge amounts of chemical additives to the aquaculture water during the breeding process, resulting in irreversible effects on the ecological environment (Wu et al., 2006). In recent years, outbreaks of white spot syndrome and acute hepatopancreas necrosis syndrome have caused the near extinction of L. vannamei aquaculture (Argue et al., 2002). To save the industry, considerable attention has been paid to ecological breeding (Moss et al., 2001, 2007, 2011). Of note, research has shown that microecological preparations used during the breeding of Chinese shrimp (Penaeus chinensis) can significantly improve the seed emergence rate (Li et al., 2007).

Microecological preparations are a general term for probiotics that can be used in aquaculture (Jiang et al., 2014). Bacillus and Lactobacillus bacteria are two kinds of probiotics commonly used in aquaculture and breeding (Zeng et al., 2007). Studies have shown that Bacillus can produce a variety of digestive enzymes to help animals digest and absorb nutrients, thereby contributing to their growth and survival (Hu et al., 2012; Samocha et al., 2004; Gong et al., 2012). Lactobacillus are gram-positive bacteria that produce lactic acid (Qiu et al., 2005), and in aquaculture water can reduce the ammonia nitrogen content, decompose undesirable material and effectively control water quality (Shen et al., 2004; Yang et al., 2001).

In the present study, we added Bacillus and Lactobacillus regularly during the entire breeding process to study their effects on L. vannamei seed growth and survival and impact resistance of young shrimps. This research will provide reference data for the improvement of L. vannamei aquaculture breeding.

Materials and methods

We purchased L. vannamei nauplii from the Shenzhen Global Group (China). The Bacillus preparation, containing 1.2 x l09 CFU g-1 of viable bacteria, was obtained from the Chinese Academy of Fishery Sciences South China Sea Fisheries Research Institute (Guangzhou, China). The Lactobacillus preparation (1 x 108 CFU*mL-1) was purchased from Xinhailisheng Biotechnology Co., Ltd., (Guangzhou, China) in liquid form.

Table I.- Feeding and management during the breeding of Litopenaeus vannamei.

Growth stage###Diets###Feeding time###Temperature(AdegC)###Inflation###Light intensity

Nauplii###Do not feed, using their###-###30###Microwave Shape###Avoid hard light

###own yolk nutrients

Zoea###Opening bait (unicellular###Every 3h###30###Slightly boiling###Avoid hard light

###algae, shrimp slices)

Early Mysis###Rotifers, prawn crackers###Every 4h###30###Boiling###Enhancing light

Mysis medium,###Increase investment of###Every 6h###30###High boiling###Increase brightness

larval shrimp###larva of Artemia

The nauplii of L. vannamei were cultured in indoor glass fiber buckets (300 L), each containing 200,000 larvae. The nauplii were divided into seven groups, including a control group (C), three Bacillus culture groups (Y1, Y2, Y3), and three Lactobacillus culture groups (R1, R2, R3). The experiments were carried out for 21 d (from 28 August to 18 September 2016). Table I shows the breeding management methods during the seeding process. Bacillus were added to Y1 (10 ppm), Y2 (15 ppm) and Y3 (20 ppm) on days 0, 1, 6, 11 and 16. Lactobacillus bacteria were added to R1 (20 ppm), R2 (25 ppm) and R3 (30 ppm) on the same days. No probiotics were added to the C group. Seawater (temperature from 27.9AdegC to 31.1AdegC, salinity from 30.0 PSU to 32.0 PSU, pH from 7.5 to 8.0) was unchanged during the experiment, with continuous aeration for 24 h and dissolved oxygen maintained above 5.0 mg*L-1 (Table I).

After the nauplii metamorphized to mysis stage, water samples (1 L) were collected from the upper, center and bottom of the barrels. After further metamorphosis, those mysis that turned into larval shrimp were collect. At the end of the 21-d experiment, all larvae in each barrel were weighed (W1, corrected to 0.001g), after which 100 shrimp seeds were randomly collected from each barrel and again weighed (W2). The average weight of the individual shrimp (W) and the survival rate of the larvae (SR) were then determined using the following formulae:

W = W2 / 100

SR = [(W1 / W) / 20000] x 100%

Resistance test of shrimp seeds

Fresh water survival measurement: 100 shrimp seeds were used for each group and transferred to fresh water, with the number of live shrimp seeds after 30 min and 60 min then counted. Survival rate after temperature change: 100 shrimp seeds were used for each group and were transferred to water with a temperature of 5AdegC and the same salinity; after 2 min the shrimp were removed and transferred into the original culture barrels. The number of shrimp were then counted after 15 min.

Statistical analyses

The data were analyzed using SPSS 17.0 for Windows software. One-way ANOVA was used to compare the differences between periods in the same group and t-tests were used to determine the differences between groups in the same period. Percentage data were analyzed by square root sine transformation, with the results shown as per the original data. We considered P 0.05) and no significant differences between Y2, Y3 and R3 (P > 0.05). The body weights of the Y2 larvae were 27.18%, 11.97%, 27.18% and 23.58% higher than those in groups C, Y1, R1 and R2 (P 0.05).

Effect of different probiotics on L. vannamei survival at all metamorphic stages

After the L. vannamei shrimp seeds metamorphosed to the mysis stage, the survival rate of all groups ranged from 70%-80% and exhibited no significant differences. However, after the shrimp seeds transformed to the larval stage, the survival rates of the experimental groups were markedly higher than that of the control group (P > 0.05). The survival rate of the shrimp larvae in R3 was 10.45% and 5.21% higher than that of the C and Y1 groups, respectively (P 0.05). At the twenty-first day of the experiment, R3 demonstrated the highest survival rate and, except for R2, was significantly higher than that in groups C, Y1, Y2, Y3 and R1 (34.86%, 21.49%, 17.60%, 15.75% and 15.75%, respectively; P 0.05).

Effect of different probiotics on stress resistance of L. vannamei seeds

After the shrimp seeds were placed in fresh water for 30 min, the survival rate for all groups was 100% (Fig. 3) and there were no significant differences (P > 0.05). After 60 min, however, the survival rates of the experimental groups were significantly higher than that of the control group (P 0.05). When the temperature changed abruptly (Fig. 4), the survival rates of groups Y1, Y2, Y3, R1, R2 and R3 were 9.29%, 9.84%, 10.38%, 8.74%, 9.29% and 10.38% higher, respectively, than that of the control group (P 0.05).


Effect of microecological preparations on growth performance of shrimp seeds

Microecological preparations have been applied within the Chinese aquaculture industry since the 1990s (Jiang et al., 2014). Microecological preparations are mainly comprised of either species that directly act on the breeding animals to promote growth and improve digestion functions and the utilization of feed, or species that act on the water medium to improve the environment to promote survival, ecological breeding and healthy farming (Zeng et al., 2007). Studies have indicated that Bacillus can improve the activity of digestive enzymes in aquatic animals, increase the digestibility of animal feed and improve the conversion rate of nutrients (Amaya et al., 2007). Lactobacillus bacteria can reduce the content of ammonia nitrogen in water, decompose detritus and organic matter in bottom water and effectively control water quality (Li et al., 2001; Jiang et al., 2014).

Our experimental results showed that by regularly adding Bacillus and Lactobacillus to the aquaculture water, the weights of the L. vannamei seeds improved, albeit to different degrees, in accordance with previous research on Chinese white shrimp (Fenneropenaeus chinensis; Wu et al., 2006). The body weights of groups Y2, Y3 and R3 were significantly higher than the weights of groups C, R1 and R2. Bacillus can break down organic material and improve rearing water (Yang et al., 2001). In this experiment, floc-like material was observed in the Bacillus treatment groups. As larvae fed on these Bacillus, adding the correct amount significantly improved the body mass of emerging seeds. Lactobacillus bacteria regulate water quality, whereby they reproduce to occupy niches and inhibit the growth of harmful bacteria (Samocha et al., 2004).

The R1 and R2 experimental groups, with relatively small amounts of Lactobacillus, did not display significant catalytic roles in improving the body mass of the emerging shrimp seeds. Therefore, we next isolated and identified the bacterial species in the water of the Lactobacillus experiment group to determine the mechanism of Lactobacillus bacteria for improving the aquaculture environment; in addition, the microecological preparations were mixed and used in the nursery process to analyze and evaluate their effects.

Different probiotics affected the survival rate of all metamorphic stages of L. vannamei. After the shrimp underwent metamorphosis from zoea stage to mysis stage larvae, there were no significant differences between the experimental and control groups in the metamorphosis rate, consistent with previous research (Yang et al., 2011). This was likely due to the relatively good water quality and suitable bait during the early period of the experiment, which allowed healthy shrimp seeds to metamorphose to the mysis stage. After the mysis stage larvae metamorphosed into juvenile shrimp, there was a certain degree of deterioration in water quality due to the accumulation and decomposition of high-protein residual bait and feces.

As probiotics can adjust water quality to a certain extent, the metamorphosis rate of juvenile shrimp in the experimental group was significantly higher than that of the control group, with R3 demonstrating the highest rate and Y1 (with the smallest amount of Bacillus) demonstrating an obviously lower rate. This was possibly due to the Lactobacillus bacteria possessing better regulative ability compared with the Bacillus bacteria, or that the propagation speed of Lactobacillus bacteria was faster than that of Bacillus in the nursery waters. Thus, we next measured seeding water quality indicators and total number of different bacteria.

Effect of microecological preparations on the resistance of L. vannamei seeds

Stress resistance, also referred to anti-stress, is an index of the ability of an animal to adapt to sudden changes in environment indicators. When faced with a rapid increase or decrease in temperature or salinity, unexpected bacterial infection outbreaks, or sudden changes in food availability, animals will experience stress. Stress resistance is not only related to genetic factors but also to an animal's health status (Yang et al., 2001; Peng et al., 2004). Water temperature and salinity changes are common stress factors in shrimp breeding (Yang et al., 2001). Therefore, we studied stress resistance in the shrimp seeds based on these factors. Results showed that almost no shrimp died under low stress conditions (salinity of 0 in fresh water for 30 min). These findings indicate that L. vannamei shrimp seeds have a broad ecological amplitude, and can withstand and survive salinity changes (from normal salinity to a salinity of 0) for at least 30 min.

However, with the extension of time in fresh water, considerable differences were observed among the control and experimental groups. Specifically, the survival rate of larvae in the control group was significantly lower than that in the experimental groups (P 0.05). The temperature experiments also obtained the same results. Our study further showed that in the L. vannamei breeding process, only a small amount of probiotics was required to significantly improve the resistance of shrimp. Here, the Bacillus promoted the digestive function of shrimp as well as the protease activity of the seeds, whereas the Lactobacillus improved the water environment of the nursery. Thus, the two probiotics improved and facilitated the physical fitness of the shrimp seeds in different ways.

Based on our research results, probiotics should be added during the breeding process of L. vannamei to enhance the resilience of shrimp seeds and reduce their mortality rate during transportation or after seeding into aquaculture ponds.

Considering our experimental findings in regard to actual production, the addition of microecological preparations during the breeding stage can increase the survival rate and individual weight of L. vannamei seeds, and also enhance the constitution and stress resistance of shrimp seeds. Furthermore, the amount of Bacillus should be greater than that of Lactobacillus (calculated by living bacteria). The spraying cycle and amount of microecological preparations are closely related to factors such as seeding pool load and water quality. Therefore, application of microecological preparations should be flexibly adjusted according to the effective microbial content, physiological activity and ecological characteristics of the various preparations to achieve the best results.


This research was supported by the Special Scientific Research Pilot Project of Sanya (2014KS03).

Statement of conflict of interest

The authors declare that there is no conflict of interests.


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Article Details
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Author:Jiang, Song; Zhou, Fa-Lin; Huang, Jian-Hua; Yang, Qi-Bin; Yang, Li-Shi
Publication:Pakistan Journal of Zoology
Article Type:Report
Geographic Code:9CHIN
Date:Jun 30, 2019
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