Phytogenic feed additive, PFA; Pacific white shrimp; Feed utilization; Fish meal sparing; growth performance; mid-gut structure
- Xiao-ling Huang∆, Laboratory of Fish Nutrition, School of Marine Sciences, Ningbo University, Ningbo
- Rui A. Gonçalves* ∆, BIOMIN Holding GmbH, Erber Campus 1, 3131 Getzersdorf, Austria
- Ming-hong Xia, Laboratory of Fish Nutrition, School of Marine Sciences, Ningbo University, Ningbo
- Yan Zhang, BIOMIN Feed Additive (Shanghai) Co. Ltd
- Gonçalo A. Santos, BIOMIN Holding GmbH, Erber Campus 1, 3131 Getzersdorf, Austria
- Pedro Encarnaçao, BIOMIN Singapore Pte Ltd, 3791 Jalan Bukit Merah #08-08, E-Centre@Redhill, Singapore 159471
- Qi-cun Zhou, Laboratory of Fish Nutrition, School of Marine Sciences, Ningbo University, Ningbo
∆Both authors contributed equally to this manuscript
Pacific white leg shrimp, Litopenaeus vannamei, has become a worldwide crustacean species in aquaculture. It is popular among farmers due to its great economic value, rapid growth rate, and tolerance to a wide range of salinity and temperature [1, 2]. Shrimp farming is thus an expanding industry, which is exerting an increasing demand on the availability of formulated feeds and the raw materials required for their manufacture. Pacific white shrimp feed is highly dependent on fish meal. Fish meal is the most important protein source for aquaculture industry due to its palatability and quality, but as a limited food resource there is serious concern on its long-term availability for use in aquaculture diets [3, 4]. The quantity of fish meal used by the aquaculture feed sector has increased as a response to the high demand for this ingredient with great impact on its price. Significant progress has been made over the past decade in reducing levels of fish meal in commercial diets for farmed aquatic animals. However, fish meal replacement by alternative protein sources such as vegetable proteins can affect the production performance and physiological competence. Thus, it is a challenge to find a new way to reduce fish meal levels in shrimp feeds without compromising performance and feed efficiency . Phytogenic feed additives (PFAs) are plant derived products which are added to the feed in order to improve animal performance. Plant essential oils have shown to exert multiple effects on the animal, such as stimulate appetite, antimicrobial action and direct reduction of gut bacteria, stimulation of gastric juices, enhance immune-system, anti-inflammatory and anti-oxidant properties [6, 7, 8, 9, 10]. However it seems that the effect of PFAs greatly depends on the species. Recently, PFAs have been examined in channel catfish  and rainbow trout Oncorhynchus mykiss (Walbaum) [12, 13] and results show improvements in weight gain, FCR, and immunity to disease. But in the other hand Peterson et al.,  did not found any improvement in growth performance or disease resistance with the addition of essential oils that contained thymol and carvacrol these aromatic compounds on channel catfish diets. A possible mode of action of the PFA supplemented and that will was tested at this experiment, is that it improves intestinal conditions and promotes nutrients absorption through an antimicrobial effect. Essential oils or some of their active constituents are indeed effective against a great variety of bacteria [15, 16, 17]. Giannenas et al.  showed that the supplementation of diets with carvacrol or thymol improved feed efficiency and decreased total anaerobe counts compared to control diets. The relation between intestinal microorganisms and intestinal structure is complex, but it is known to have an impact in nutrient absorption . However, the antimicrobial mechanism of essential oil components is not well defined, and this may be due to the great number of constituents of such products. Typical lipophilic substances pass through the cell wall and cytoplasmic membrane of bacteria and damage them, which leads to the leakage of macromolecules and to the subsequent lysis of bacteria [18, 19, 20, 21, 6]. Essential oils can also coagulate the cytoplasm  and damage lipids and proteins .
Due to their effects on improving feed efficiency, PFAs could be an important tool to reduce feed cost in the context of high priced feed ingredients such as fish meal (FM). The objective of present study was to evaluate the nutrient sparing effect of a commercial matrix-encapsulated essential oil (EO) product (Digestarom® P.E.P. MGE) in Pacific white shrimp feed. The product was tested alone under controlled, indoor conditions. The study evaluated the effect of the PFA on growth performance, feed utilization and gut intestine ultrastructure in Pacific white shrimp, fed diets with reduced fish meal.
Table 1: Formulation and proximate composition of the experimental diet (dry matter)
2.1 Growth performance and feed utilization
Growth performance and feed utilization of Pacific white shrimp fed the experimental diets was numerically worsened with reduction of fish meal level in the diet; however, this reduction was not statistically significant (p>0.05) among treatments (Table 2). The control diet, with higher content of fish meal (FM25) presented the best performance; however in low content fish meal diets, diets supplemented with Digestarom® P.E.P. MGE (FM22 + Digestarom® P.E.P. MGE and FM19 + Digestarom® P.E.P. MGE ) showed better performance in comparison with non-supplemented ones (FM22 and FM19).
Weight gain during the experimental period (data not shown)showed no significant differences (p=0.113) when comparing treatment groups with the control group (FM25); however when compared with the respective control diet, shrimp fed diets supplemented with Digestarom® P.E.P. MGE presented increased weight gains. Regarding specific growth rate (SGR; p=0.117) and feed conversion rate (FCR; p=0.094), no significant differences were found, with FM25 presenting the best growth results. In terms of the diets with reduced fish meal, diets supplemented with Digestarom® P.E.P. MGE improved shrimp SGR and FCR numerally (Table 2).
Table 2: Growth performance parameters of juvenile Pacific white shrimp fed different experimental diets.
PER- Protein Efficiency Ratio; HSI – Hepatosomatic Index. Data represent mean ± S.D. of five replicates. No significant differences were found on these parameters (P>0.05).
2.2 Proximate composition of muscle and whole body
Proximate composition of muscle and whole body of shrimp fed the experimental diets are presented in Table 3. Moisture, lipid, protein and ash contents in whole body and muscle were not significantly influenced by the diets supplemented with the EO combination (p>0.05).
Table 3: Whole body and muscle composition of juvenile Pacific white shrimp fed different experimental diets.
Data represent mean ± S.D. of five replicates. No significant differences were found on these parameters (p>0.05).
2.3 Haematological parameters
Haematological characteristics of juvenile Pacific white shrimp fed the experimental diets are shown in Table 4. Digestarom® P.E.P. MGE supplementation did not significantly affect total protein, glucose, triacylglycerol and cholesterol concentrations in the hemolymph. Shrimp fed the diet containing higher levels of fish meal presented higher glucose, triacylglycerol and cholesterol concentrations than those fed diets containing lower levels of fish meal diets. Shrimp fed the FM22 + Digestarom® P.E.P. MGE had the lowest total protein level in serum.
Table 4: Haematological characteristics of juvenile Pacific white shrimp fed different experimental diets.
Data represent mean ± S.D. of five replicates. No significant differences were found on these parameters (p<0.05).
2.4 Haematological enzyme activities
The effects of dietary treatments on ALT, AST, AKP and LDH activities in the serum are presented in Table 5. These activities were not significantly influenced by the dietary treatments (p>0.05). Shrimp fed the diet FM25 had higher ALT and AST than those fed the other diets. However there are some interesting tendencies: all five parameters analysed (ALT, AST, AST/ALT, AKP and LDH) were numerically decreased when the amount of fish meal in the diets was reduced. On treatment FM22, with the exception of AKP, there was an increase of enzyme activities’ values on the supplemented diets. For diet FM19 this tendency was not clearly observed.
Table 5: Haematological enzyme activities of juvenile Pacific white shrimp fed different experimental diets.
ALT: Alanine aminotransferase; AST: Aspartate transaminase; AKP: Alkaline phosphatase; LDH: Lactate dehydrogenase. Data represent mean ± S.D. of five replicates. No significant differences were found on these parameters (P<0.05).
2.5 Structure of the gastrointestinal tract
Shrimp gut tissue structures analysed through electronic microscope are presented in Figure 1. Shrimp fed the Control diet had better structured and higher number of microvilli in the mid-gut than those fed the other diets. However in shrimp fed low fish meal diets, shrimp supplemented with Digestarom® P.E.P. MGE had better structured and higher number of microvilli in midgut than those fed non-supplemented diets.
Figure 1: Effect of Digestarom® P.E.P. MGE supplementation on ultrastructure of mid-gut tissue (microvilli) observed by transmission electron microscope.
The replacement of fish meal by plant proteins in attempt to reduce feed costs and reduce the dependency on fish meal negatively affect feed efficiency and gut health, in the current study. Plant raw materials are less digestible and in addition can cause negative effects directly on the gastrointestinal tract. The presence of undigested nitrogenous compounds in the intestine favours the formation of ammonia and biogenic amines by the intestinal microbiota, which results in inflammatory processes and accelerated turnover of the intestinal tissue, leading to poorer performance . The results obtained in this study, did not allow us to make clear conclusions, nonetheless, results indicate important improvements on measured parameters when using Digestarom® P.E.P. MGE. The overall results from this experiment show that shrimp receiving the 25.0% fish meal diet (FM25) present the best haematological characteristics and the best growth performance. Digestarom® P.E.P. MGE supplementation led to improved growth performance and haematological values in diets containing lower fish meal concentrations (22.0% an 19.0%). Analysis of gut tissue structure showed, that shrimp microvilli were neater and in higher number when supplemented with Digestarom® P.E.P. MGE compared to the respective control (shrimp receiving diets with lower amounts of fish meal but without the supplementation of a PFA). Similar results were observed in other species, such as gilthead seabream  and channel catfish . Yet available literature data on the efficacy of dietary essential oils on the zootechnical performance of shrimp is extremely scarce. Zheng et al  indicated that the supplementation of feeds with carvacrol, a combination of carvacrol and thymol and oregano essential oil enhanced weight gain. Moreover, the use of an essential oil blend (anise, citrus and oregano) was found to enhance weight gain of channel catfish and this effect was mainly associated to a higher feed intake .
In this study, WG, FCR and PER decreased as well as the distance and number of microvilli in intestine, with the reduction of fish meal levels, mainly on the diets without EO supplementation. This may suggest that the replacement of fish meal by soybean meal can cause enteritis due to the excess of anti-nutritive components. It is well described in literature that higher inclusion levels of full-fat and solvent-extracted soybean meal not only causes reduced weight gain and feed efficiency [26, 27, 5, 28] and led to morphological changes of the distal intestinal epithelium [29, 30]. Some of the inherent anti-nutritive components in soybeans have also been implicated in reduced growth and/or impaired digestive functions, such as soybean trypsin inhibitor activity , saponins  and lectins . Soybean meal can also increase permeability of distal intestinal epithelium for nutrients and the capacity of this region to absorb nutrients is also reduced . Baeverfjord and Krogdahl  reported that the enteritis induced by dietary soybean meal was classified as non-infectious sub-acute enteritis. In this experiment, for the same fish meal level, diet supplementation with Digestarom® P.E.P. MGE improved WG, FCR, PER and distance/number of microvilli in the intestine. It is well known that the digestive tract is divided into three distinct regions, according to their relative importance in all crustaceans. The foregut and hindgut have a chitinous lining and do not play an important role in digestive processes ; therefore, the improvement of microvilli is important for nutrients absorption.
Changes in enzyme activity are also a good parameter to assess fish health [36, 9]. ALT and AST are intracellular aminotransferases which catalyze the transfer of amino groups from α-aminoacids to α-ketoacids. ALT is an enzyme involved in amino acid metabolism and during the process of tissue destruction, this intracellular enzyme is released to the circulating blood. Elevated serum ALT levels are a symptom of a variety of liver diseases . Thus, ALT can be used as a reasonable specific indicator of liver disease. AST is an enzyme that catalyzes the transfer of an amino group between L-aspartate and α-ketoglutarate to form oxaloacetate and L-glutamate. This enzyme is not specific for liver disease, however, to some extend it can be used to indicate liver disorders . LDH is an oxidoreductase that catalyzes the interconversion of lactate and pyruvate. When tissues get infected or injured, they release LDH into the bloodstream. As such, this enzyme is frequently used to evaluate tissue or cell damage .
The haematological enzyme results of the study showed a reduction of ALT when fish meal in the diets was reduced. This fact can be derived from the lower turnover rate in liver cells due to the less digestible protein in diets FM22 and FM19. On treatment FM22, with the exception of AKP, there was an increase of enzyme values on supplemented diets. The variations around the mean values of the measured parameters did not allow us to make clear conclusions; however haematological enzyme activity did not reflect tissue or cell damage due to the use of Digestarom® P.E.P. MGE.
It is clear that the replacement of fish meal by plant protein sources suggested an negative impacts on SGR, although not significant levels were found. This is probably because plant protein is less digestible, which has direct negative effects on the gastrointestinal tract. However, the use of Digestarom® P.E.P. MGE suggest an improvement in the SGR in treatments FM22 and FM19 which had some fish meal replaced by plant protein. Unlike expected, the 19.0% fish meal diet supplemented with Digestarom® P.E.P. MGE had better SGR than the 22.0% fish meal diet. Theoretically, the use of less digestible plant raw material increases the presence of undigested nitrogenous compounds in the intestine which induces intestinal microbiota to produce ammonia and biogenic amines. These compounds are toxic and consequently can lead to the imbalance of the intestine, resulting in inflammatory processes and accelerated turnover of the intestinal tissue and subsequently to poorer performance.
The replacement of fish meal by plant proteins had a negative influence in feed conversion ratio as the FCR increased with the reduction of fish meal in the diets. However, the supplementation of low fish meal diets with Digestarom® P.E.P. MGE suggest an improvement in FCR values when compared with non-supplemented diets. Survival index was not significantly affected by the treatments (p>0.05).
Overall results suggested that Digestarom® P.E.P. MGE can improve the growth performance of white leg shrimp even in low fish meal diets. As a natural growth promoter, it presumably acts on the intestinal microbiota and lead to improved animal performance.
The results of this experiment suggest that the modulation of intestinal microflora can positively influence animal performance and that essential oil blends have the potential to be used in this goal. Further studies are needed to understand the exact mode of action of essential oils and their potential use as alternative growth promoters in shrimp.
4. Materials and Methods
4.1 Diet preparation
Five diets were formulated to contain approximately 40.0% crude protein and 8.5% crude lipid on a dry-matter basis. Fish meal, soybean meal (SBM), peanut meal and brewer's yeast were used as protein sources; soybean oil, fish oil and soy lecithin were used as lipid sources; wheat flour was used as a carbohydrate source (Table 1). Three levels of fish meal as marine-derived protein source were used. The control diet contained 25.0% fish meal (FM25), the highest fish meal level used. The fish meal level was then reduced to 22.0% (FM22) and 19.0% (FM19) by substitution with SBM and peanut meal. An essential oil combination product (Digestarom® P.E.P. MGE; BIOMIN Holding GmbH, Austria) was then supplemented to the diets with reduced fish meal content: FM22 + Digestarom® P.E.P. MGE and FM19 + Digestarom® P.E.P. MGE at a dosage of 200 g/tonne of feed. These last two fish meal levels were also evaluated without any EO supplementation. Digestarom® P.E.P. MGE it’s a matrix-encapsulated phytogenic additive characterized by carvacrol, thymol, and limonene. Dietary ingredients were ground through an 80 mm-mesh and weighed (±0.01 g). All dry ingredients were thoroughly mixed to homogeneity in a Hobart-type mixer. Lipids and 1500 mL distilled water were then added and thoroughly mixed. Cold-extruded pellets (1.0 mm and 1.5 mm in diameter, 3 mm in length) were produced and air dried (20-27 °C) to approximately 10% moisture, then sealed in vacuum-packed bags and stored frozen (-20 °C) prior to use in the feeding trial.
4.2 Shrimp source and experimental design
Juvenile Pacific white shrimp were obtained from the shrimp hatchery of Guangdong Evergreen Group (Zhanjiang, China). Prior to the experiment, the shrimp were acclimated to laboratory conditions and fed with a commercial diet (40.0% crude protein, 8.0% crude lipid; Guangdong Evergreen Group, Zhanjiang, China) in 1000 L cylindrical fiberglass tanks for 2 weeks. At the beginning of the experiment, 40 shrimps (initial weight of approximately 0.33±0.02 g) were stocked randomly and sorted into a 500 L cylindrical fiberglass tank with lentic system. Each diet was randomly assigned to five replicate groups. All groups were fed by hand at 6-10% of body weight per day divided into four daily feedings at 7:00, 12:00, 17:00 and 21:00 h. The daily feeding amount was calculated to assure apparent satiation without overfeeding, and the feeding rate was adjusted every two weeks after weighing the biomass in each tank. Dead shrimp were removed, weighed and their weight was recorded. Each tank was cleaned biweekly. At the end of the trial, shrimp from each tank were removed and weighed as a group. The duration of the feeding trial was 8 weeks.
Each tank was supplied with continuous aeration by air stones to maintain dissolved oxygen levels at, or near, saturation. During the experimental period, dissolved oxygen was not less than 6.0 mg/L and ammonia nitrogen content was lower than 0.05 mg/L. The temperature was 26.5±1.2 °C, salinity was 27.5±1.1 g/L and pH was 8.1±0.1.
4.3 Sample collection techniques and analyses
At the beginning of the feeding trial, 120 shrimp were randomly sampled and frozen (-20 °C) for analysis of initial proximate composition. At the end of the 8-week feeding trial, shrimp in each tank were weighed and counted. Six shrimp per tank were randomly sampled for proximate composition analysis of whole body. Another ten shrimp from each tank were anesthetized with tricaine methane sulfonate (MS-222), and hemolymph samples of approximately 1.0 mL were collected from the pericardial cavity by 1.0 mL syringe. The samples were transferred to 1.5 mL Eppendorf tubes, centrifuged at 4 °C, 5000 rpm for 10 min, and the supernatant was then collected, packaged and stored at -80 °C until analysis of enzyme activity.
The moisture, crude protein, crude lipid and ash contents in the diets and whole bodies of the shrimp were determined according to the procedures recommended by the Association of Official Analytical Chemists . Moisture was determined by drying the samples to a constant weight at 105 °C. Crude protein contents (N × 6.25) were determined by the Kjeldahl method following acid digestion with an auto-digester (FOSS, Tecator, Hoganas, Sweden), crude lipid content via the ether extraction method, using a Soxtec System HT (Soxtec System HT6, Tecator, Sweden) and ash contents in a muffle furnace run at 550 °C for 8 h.
The hemolymph was determined within 24 h of storage at 4 °C, and then the serum was collected by centrifuging hemolymph at 5,000 rpm for 10 min. The total protein, glucose, triacylglycerol and cholesterol contents in the hemolymph were assayed using an automatic blood analyser (Hitachi 7170A, Japan). Alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (AKP) and lactate dehydrogenase (LDH) in hemolymph were analysed using an automatic blood analyser (Hitachi 7170A, Japan) from a clinical laboratory in Ningbo University Hospital. The effects of the supplementation on ultrastructure of mid-gut tissue (microvilli) were observed by transmission electron microscope.
4.4 Calculations and statistical analysis
The following parameters were calculated: Percent weight gain (WG, %) = 100×(Wt−Wi)/Wi [Wt is the final body weight (g); Wi is the initial body weight (g)]; Specific growth rate (SGR, %/day) = 100×(Ln Wt−Ln Wi)/t [t is the experimental duration in days; Ln is the natural logarithm]; Protein efficiency ratio (PER) = weight gain (g, wet weight)/protein intake (g, dry weight); Feed conversion ratio (FCR) = weight gain (g, wet weight)/feed consumed (g, dry weight); Survival (%) = 100×(final number of shrimp)/(initial number of shrimp); Hepatosomatic index (HSI, %) = hepatosomatic weight×100/wet weight.
Results are shown as means ± standard deviation. All data generated from the trial were verified for data normality using a Shapfro-Wilks test. Then, data was analysed using the non-parametric Kruskal–Wallis test to explore differences between treatments. A significance level of 95% (α=0.05) was set for all the tests. All statistical analyses were performed using SPSS 19.0 (SPSS, IL USA).
The study resulted in collaboration between BIOMIN Holding GmbH, Erber Campus 1, 3131 Getzersdorf, Austria and the Laboratory of Fish Nutrition, School of Marine Sciences, Ningbo University, Ningbo 315211.
6. Author Contributions
Gonçalo A. Santos, Pedro Encarnaçao, Rui A. Gonçalves and Qi-cun Zhou the scientific leaders and the project supervisors; designed the experiment presented in this paper. Xiao-ling Huang, Ming-hong Xia and Yan Zhang performed the experiments. Rui A. Gonçalves analysed the results and wrote the manuscript with support of Gonçalo A. Santos, Pedro Encarnaçao and Qi-cun Zhou.
7. Conflicts of Interest
The authors declare no conflict of interest.