Veterinary Microbiology 182 (2016) 44–49

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Effect of poly-b-hydroxybutyrate on growth and disease resistance of

Nile tilapia Oreochromis niloticus juveniles
Magdalena Lenny Situmoranga,b , Peter De Schryvera,* , Kristof Dierckensa , Peter Bossiera
a
Laboratory of Aquaculture & Artemia Reference Center, Department of Animal Production, Ghent University, Rozier 44, 9000 Ghent, Belgium
b
School of Life Sciences and Technology, Bandung Institute of Technology, Jalan Ganesha 10, Bandung, Indonesia

A R T I C L E I N F O A B S T R A C T

Article history: The growth promoting effect of the bacterial storage compound poly-b-hydroxybutyrate (PHB) has been
Received 3 June 2015 studied for young fish of high trophic level (European sea bass) and intermediate trophic level (Siberian
Received in revised form 20 October 2015 sturgeon). Here, the effect of PHB on growth, digestive enzyme activities, body composition and diseases
Accepted 26 October 2015
resistance of juvenile Nile tilapia (Oreochromis niloticus) of low trophic level was investigated. Although
dietary PHB supplementation (5, 25 and 50 g PHB kg
1 formulated semi-purified diet) during 28 days
Keywords: resulted in a trend of increased weight gain, there was no significant difference in the mean final body
Poly-b-hydroxybutyrate
weight (258–284 mg) when compared to the fish from the control group (on average 218 mg). Lipase
Nile tilapia
Digestive enzyme activity
activity increased significantly with about 20–40% by the supplementation of PHB in the diet, which may
Lipid have led to the significant increase in total lipid content with about 10% in the PHB treatment groups.
Disease However, the profile of total (n-6) fatty acids (FAs), total monounsaturated FAs and total saturated FAs
relative to the total lipid was similar among various PHB treatments. An additional challenge test on
gnotobiotic Nile tilapia larvae using the pathogen Edwardsiella ictaluri gly09R showed that feeding
challenged larvae with PHB-enriched Artemia nauplii resulted in a 20% higher survival as compared to the
challenged control larvae. Overall, it is suggested that the trend of increased body weight gain resulted
from intestinal lipid digestion, absorption and deposition and that PHB is effective as an antimicrobial
agent for application in Nile tilapia larviculture.
ã 2015 Elsevier B.V. All rights reserved.

1. Introduction Several studies have confirmed the protective effect of PHB

against bacterial infections in crustaceans (Defoirdt et al., 2007; Sui
Short-chain fatty acids (SCFAs) have been suggested as an et al., 2012; Thai et al., 2014), but interestingly enough an increased
alternative to antibiotics to control diseases in aquaculture culture performance in crustaceans has also been observed. For
(Defoirdt et al., 2009). Their main limitation, however, is their example, Nhan et al. (2010) and Sui et al. (2012) found an increased
polar nature making them highly soluble and easy to diffuse in the growth performance and survival in larval giant freshwater prawn
culture water. This results in the risk for a low uptake efficiency of (Macrobrachium rosenbergii) and larval Chinese mitten crab
the SCFAs by the animals when formulated in a diet and thus (Eriocheir sinensis), respectively, upon the use of crystalline PHB
demands for large doses in order to be effective at the animal level (i.e. PHB extracted from the bacterial cell) in the diet. Most
(Defoirdt et al., 2011). A solution has been provided in the form of recently, Thai et al. (2014) described that the supplementation of
the bacterial storage compound PHB, the polymer of the SCFA PHB in amorphous form (i.e. PHB still contained within a bacterial
b-hydroxybutyrate (b-HB). This carbon reserve and intracellular cell) to the diet was effective to boost the growth and development
energy source for different species of bacteria (De Schryver et al., of M. rosenbergii larvae. The beneficial effects of dietary PHB
2010; Defoirdt et al., 2011; Tokiwa and Calabia, 2007) is water supplementation on the growth performance of fish has only been
insoluble and biologically degrades into b-HB upon entrance in the investigated in a limited number of cases. In carnivorous European
gastrointestinal tract (Anderson and Dawes, 1990; Doyle et al., sea bass (Dicentrarchus labrax) juveniles a growth promoting effect
1991). was observed at 20 and 50 g PHB kg
1 diet (De Schryver et al., 2010)
while for omnivorous Siberian sturgeon (Acipenser baerii) finger-
lings no significant increase in weight was observed resulting from
dietary PHB supplementation (Najdegerami et al., 2012). The
protective effect of PHB against pathogenic diseases has, as far as
* Corresponding author. Fax: +32 9 2644193.
E-mail address: [email protected] (P. De Schryver).
known, not yet been investigated for fish.

http://dx.doi.org/10.1016/j.vetmic.2015.10.024
0378-1135/ ã 2015 Elsevier B.V. All rights reserved.
 M.L. Situmorang et al. / Veterinary Microbiology 182 (2016) 44–49 45

In this study, the effect of crystalline PHB administered via a experimental diets were the basal diet supplemented with three
semi-purified diet to Nile tilapia (Oreochromis niloticus) at the early levels of crystalline PHB (5, 25, and 50 g kg
1 diet; Goodfellow
juvenile stage was investigated in terms of survival and growth Cambridge Limited, Huntingdon, England), at the expense of
performance, the digestive enzyme activities and body lipid a-cellulose. The moist mixture was extruded through a 3-mm
composition. Additionally, the protective effect of amorphous PHB diameter meat grinder (Hobart Corp., Troy, Idaho, USA). The
supplied through Artemia to larval Nile tilapia challenged with the resulting moist pellets were air-dried at room temperature to a
bacterial pathogen Edwardsiella ictaluri gly09R was investigated. moisture content of about 100 g kg
1. Pellets were ground into
small pieces, sieved to obtain appropriate sizes (300–500 mm) and
2. Materials and methods stored at
20  C until used. The fish were fed daily during 28 days
at a fixed feeding level of 20% on fish wet body weight (BW) (Ng
2.1. Effect of crystalline PHB on growth of Nile tilapia juveniles and Romano, 2013) in three equal meals given every 4 h between
09:00 and 17:00. The feed amount was adjusted daily based on
2.1.1. Experimental setting mortality and weekly based on the average weight of the fish in the
Nile tilapia juveniles were obtained from the Aquaculture and tank. Each experimental diet was tested in triplicate.
Fisheries Group (AFI) of Wageningen University and Research Center
(The Netherlands). After transport, juveniles were collected and 2.1.3. Fish survival and growth parameters
acclimated in a 50 L aquarium for 14 days prior to stocking in the Six juveniles from each replicate tank were randomly collected
experimental units. During this period, they were fed a formulated at the end of the trial minimally 14 h after the last feeding to
experimental basal diet (without PHB supplementation) three times measure the final wet BW and length. The average wet BW gain of
daily to apparent satiation (Table 1). The juveniles were in healthy the fish in a tank over the 28 days was calculated by subtracting the
condition at the end of the acclimation period as indicated by the weight of the fish sampled on the final day of the experiment with
absence of deformities and abnormal movement, the absence of the average weight of the fish as measured at the beginning of the
clinical signs of bacterial and/or fungal infection and a good appetite experiment, and then taking the average. The specific growth rate
and feeding intake. At the end of the acclimation period, fish with an (SGR) was calculated as follows:
average weight of 26.4  2.4 mg were randomly distributed in
ðlnW
lnW 0 Þ
12 rectangular aquaria of 38 L at a density of 50 fish per aquarium. SGRð% weight increase per dayÞ ¼
t
During the feeding experiment, water temperature was maintained
at 28  1  C with pH ranging from 8.2 to 8.8. NH4+, NO2
, and NO3
where W is the average BW after 28 days, W0 is the average initial
levels never exceeded 0.05 mg N L
1, 0.5 mg N L
1 and 2.5 mg N L
1, BW, and t is experimental period (28 days). The same approach was
respectively. The experiment was carried out under a 16:8 h light: used to calculate the feed conversion ratio (FCR), expressed as the
dark light regime. Each tank was supplied in flow-through mode of feed consumption over the weight increase of the fish per
dechlorinated, heated tap water at a water renewal rate of 5 L h
1 treatment. Fish survival for individual treatments was determined
(hydraulic residence time of 7.6 h). In order to prevent feed loss as the number of surviving fish at the end of experimental period
during the feeding period by water exchange, the flow through relative to the number of fish at the beginning of the experimental
system was only activated during the dark period. Water flow rate period.
was checked and adjusted daily to ensure proper water exchange.
2.1.4. Fish sampling and analyses
2.1.2. Experimental diets and feeding regime
A basal semi-purified diet (control diet) was formulated to 2.1.4.1. Assessment of digestive enzyme activities. Whole-body
contain approximately 450 g crude protein kg
1 (Balarin and homogenates were used instead of intestine homogenates to
Halfer, 1982) and 150 g lipid kg
1 (Ng and Chong, 2004) based provide enough wet sample for enzymatic assays due to the small
on the feedstuff values reported in NRC (1993) (Table 1). The PHB size of the fish. Fish samples were taken minimally 14 h after the
last feeding. Three fish from each replicate tank were randomly
sampled, euthanized with an overdose of tricaine mesylate (0.1%
Table 1 tricaine methanesulfonate, MS-222; Sigma Chemical Co., St Louis,
Formulation of the basal semi-purified diet (control diet) for Nile
USA) and stored at
80  C until homogenization. Samples were
tilapia juveniles used in this study.
homogenized on ice in 50 mM Tris–HCl buffer (pH 7.5) at a 5:1 ratio
Ingredients g kg
1 (w/w) in an electric homogenizer (Heidolph, Instruments
Carboxymethyl cellulose (Sigma) 20 Switzerland). The homogenate was centrifuged at 10,000 g for
Vitamin C (Stay-C 35% VDS) 0.6 20 min at 4  C and the supernatant collected and stored in small
L-methionine (Sigma) 5 aliquots (100–200 mL) at
80  C until the spectrophotometric
Vitamins + minerals mix (VDS) 12.5
L-lysine (Sigma) 15
assays of digestive enzyme activities. Lipase activity, trypsin
Choline chloride 1.8 activity, pepsin activity, and amylase activity were quantified
a-cellulose 50 according to the procedures of Iijima et al. (1998),Holm et al.
Corn meal (bio planet) 100 (1988), Zambonino-Infante and Cahu (1994) and Métais and Bieth
Fish herring meal (VDS) 200
(1968), respectively. All assays of digestive enzyme activities were
Corn gluten meal (Sigma) 500
Fish oil (VDS) 20 carried out in triplicate using the Bio-Rad Benchmark Plus
Soybean oil 75 microplate spectrophotometer and Falcon flat-bottom 96-well
Vitamin E (95%) 0.1 microplates (Fisher Scientific). All pH values for buffers were
Analyzed nutrient content g kg
1 DM measured at room temperature, and all reagents were purchased
DM (g kg
1) 922
Crude protein 524
from Sigma–Aldrich Chemical. Each enzyme activity was
Crude fat 146 measured in each individual fish. The total protein content was
Ash 54 measured according to the method of Bradford (1976) using bovine
Total carbohydratesa 276 serum albumin as standard. The specific activity of measured
a
Calculated as follows: total carbohydrates = 1000
(crude enzymes is expressed as unit enzyme activity per mg protein
protein + crude fat + ash). (U mg
1 protein).
46 M.L. Situmorang et al. / Veterinary Microbiology 182 (2016) 44–49

2.1.4.2. Total protein and lipid content analyses. At the end of the challenge via the live food, axenic Artemia nauplii were obtained
experiment, remaining fish from each replicate tank were pooled following the procedure of Marques et al. (2004). The axenic Artemia
(total wet weight of 2–3 g) and the total protein and lipid content in nauplii were harvested, washed using 0.2 mm filtered autoclaved
the whole body of fish from each treatment was analysed following synthetic freshwater, counted and diluted to a density of 100 nauplii
standard methods. Nitrogen content was analyzed by the Kjeldahl mL
1. The pathogen was added to the axenic Artemia nauplii at a
method (AOAC, 2000) and crude protein content was estimated by density of 108 CFU mL
1. Bacteria loaded Artemia nauplii were
multiplying the nitrogen percentage by 6.25. Lipid content in the harvested after 1 h and washed twice using 0.2 mm filtered
whole body was analyzed by extraction following the modified autoclaved synthetic freshwater, counted, and added into the
Folch method (Folch et al., 1957) illustrated in Ways and Hanahan bottles containing the fish.
(1964). Fatty acid (FA) composition was determined by gas For the fish supplied of PHB, axenic Artemia nauplii at a density
chromatography of the fatty acid methyl esters (FAME) of 100 Artemia mL
1 were enriched in a 100 mg L
1 amorphous
following the modified procedure of Lepage and Roy (1984). PHB suspension for 1 h. The amorphous PHB consisted of the
bacterium Alcaligenes eutrophus containing 70% PHB on dry weight
2.2. Effect of amorphous PHB on disease resistance of Nile tilapia and was produced according to Thai et al. (2014). The enriched
larvae Artemia were subsequently washed with 0.2 mm-filtered auto-
claved synthetic freshwater and added into the bottles.
2.2.1. Disinfection protocol to obtain axenic Nile tilapia larvae The total number of Artemia nauplii added in each bottle
Nile tilapia eggs of 3 days post fertilization (3 dpf) were (independently of the treatment) was 20 per larva. As such, the
collected from female breeders, pooled, disinfected and axenically control group fish were fed 20 axenic non-enriched Artemia nauplii
hatched as described by Situmorang et al. (2014). per larva. PHB-supplemented fish were fed 10 PHB-enriched
Artemia nauplii per larva with an extra addition of 10 axenic
2.2.2. Bacterial strain and culture conditions Artemia nauplii per larva (for unchallenged fish) or 10 pathogen-
E. ictaluri gly09R with multiple antibiotics resistance as enriched Artemia nauplii per larva (for challenged fish). The
described in Situmorang et al. (2014) was used in gnotobiotic challenged fish without PHB supplementation were fed 10 axenic
challenge tests. The strain was stored in Brain Heart Infusion (BHI) Artemia nauplii and 10 pathogen-enriched Artemia nauplii per
broth (FLUKA, Sigma–Aldrich, USA) supplemented with 20% (v/v) larva. Mortality was determined daily and dead fish were removed
glycerol at
80  C. It was then grown in BHI broth containing daily during the experiment. The experimental design for the
10 mg L
1 ampicillin, rifampicin, kanamycin, trimethoprim and bacterial challenge test was approved by the ethical committee of
gentamycin, and incubated overnight on a horizontal shaker at Ghent University under the file number EC2014/041.
160 rpm at 27  C. The bacterial suspensions were harvested by
centrifuging at 1000  g for 10 min and washed twice using 0.2 mm 2.3. Statistical analyses
filtered autoclaved synthetic freshwater (Situmorang et al., 2014).
The optical density was measured using a spectrophotometer For the growth test, normalization of the distribution of the
(Genesys 20, Thermospectronic) at a wavelength of 550 nm and the survival and final BW data were done using arcsine and log
bacterial density was estimated based on the McFarland standard transformation, respectively. Comparison of the fish survival, final
(BioMérieux, Marcy L’Etoile, France). BW and length, SGR, FCR, and digestive enzyme activities were done
using one-way analysis of variance (ANOVA). Grouping of treatments
2.2.3. Bacterial challenge test for gnotobiotic Nile tilapia larvae based on significant differences in mean values was done using a
supplied with poly-b-hydroxybutyrate (PHB) Tukey post-hoc test (0.05 level of confidence). For the comparison of
At 5 days after hatching (DAH 5), 160 axenic larvae were the cumulative mortality of fish larvae in the challenge test, data
distributed over 500 mL sterile glass bottles containing 200 mL were arcsine transformed before a one-way analysis of variance
0.2 mm filtered autoclaved synthetic freshwater at a density of (one-way ANOVA) was performed. A Duncan test was performed on
10 fish per bottle. The fish were subjected to 4 treatments with four the transformed data for multiple comparisons among means
replicates during 12 days: (0.05 level of confidence). STATISTICA statistical software (version
7.0) was used for all statistical analyses.
(1) No pathogenic challenge; no PHB supplementation (control).
(2) No pathogenic challenge; PHB supplementation. 3. Results
(3) Pathogenic challenge; no PHB supplementation.
(4) Pathogenic challenge; PHB supplementation. 3.1. Effect of crystalline PHB on growth of Nile tilapia juveniles

The pathogenic challenge was done daily during the 11 days 3.1.1. Fish survival and growth parameters
experimental period via the culture water and via the live food Following the 14 days acclimatization period during which the
(Artemia nauplii). For the challenge via the culture water bacteria fish were fed the experimental basal diet without PHB supple-
were added in the bottles at a density of 106 CFU mL
1. For the mentation, the fish readily started consuming the experimental

Table 2
Survival and growth parameters (mean  standard deviation) of Nile tilapia juveniles fed four experimental diets. No significant differences were found in the fish survival,
final body weight and length, and FCR (number of replicates per treatment = 3; number of fish sampled per replicate to determine growth parameters = 6).

Treatment Survival (%) Final body weight (mg) Final body length (cm) SGR (% BW day
1) FCR
a a a
Control 87  2 218  101 2.3  0.4 5.27  0.05a 1.0  0.3a
5 g kg
1 PHB 74  8a 284  61a 2.6  0.3a 5.39  0.07b 0.9  0.0a
25 g kg
1 PHB 86  10a 269  68a 2.5  0.4a 5.24  0.05a 0.9  0.1a
50 g kg
1 PHB 88  3a 258  48a 2.5  0.4a 5.21  0.03a 0.8  0.1a

Different superscript letters within a column denote significant differences (P  0.05).

 M.L. Situmorang et al. / Veterinary Microbiology 182 (2016) 44–49 47

Table 3
Digestive enzyme activities (mean  standard deviation) of Nile tilapia juveniles following 4 weeks of feeding with different experimental PHB-supplemented diets (number
of replicates per treatment = 3; number of fish sampled per replicate = 3).

Treatment Digestive enzyme activities (U mg protein
1)

Lipase Trypsin Amylase Pepsin

Control 0.021  0.003a 0.035  0.017a 0.457  0.039a 0.253  0.010a
5 g kg
1 PHB 0.025  0.004ab 0.032  0.017a 0.533  0.041a 0.261  0.021a
25 g kg
1 PHB 0.029  0.004b 0.033  0.014a 0.489  0.042a 0.272  0.014a
50 g kg
1 PHB 0.029  0.001b 0.047  0.016a 0.547  0.043a 0.245  0.022a

Activities are expressed as follows: Lipase as mmole of substrate hydrolysed min
1 mg
1 protein; Trypsin activity as mmole of BAPNA hydrolysed min
1 mg
1 protein;
Amylase mg starch hydrolysed min
1 mg
1 protein; Pepsin activity as mmole of tyrosine released min
1 mg
1 protein. Different superscript letters within a column denote
significant differences (P  0.05).

diets and no clear changes in feeding response were observed saturated and monounsaturated FAs were significantly higher in
when the fish were provided with feed. At the end of the feeding the 5 g kg
1 and 25 g kg
1 PHB treatment groups as compared to
trial, mortality had occurred in each treatment due to aggressive the control treatment, while a significantly higher content of total
behavior of some fish in each tank. This mortality was slightly (n-6) FAs was observed in the 50 g kg
1 PHB treatment group.
higher in the 5 g kg
1 treatment but there was no significant However, PHB did not significantly influence the lipid composition
difference in survival between the different treatments (Table 2). as similar values were observed among treatments when the FAs
After 28 days of feeding, a trend of increased final BW and length were expressed as percentage on the total lipid content. PHB had
could be observed for the fish fed with PHB although there were no no significant influence on content and composition of C20:5n3
significant differences among all treatments. Only at a dose of 5 g (eicosapentaenoic acid, EPA), C22:6n3 (docosahexaenoic acid,
PHB kg
1 diet the SGR was higher than for the control treatment DHA), total (n-3) FAs, nor the (n-6)/(n-3) ratio.
(Table 2). The FCR- expressing the amount of feed dry matter
needed per unit of fish weight gain - was not significantly different 3.2. Effect of PHB on disease resistance of Nile tilapia larvae
for the fish from the four experimental diet treatments.
The protective effect of PHB for sterile Nile tilapia larvae
3.1.2. Digestive enzyme activities challenged with pathogenic E. ictaluri gly09 was assessed. In all
After 28 days of feeding with the experimental diets, no treatments, larval mortality was first observed on the 6th day after
significant differences were observed for trypsin, amylase and challenge and no significant differences were observed between
pepsin activity among the various treatments (Table 3). Feeding the treatments until 9 days after challenge (Table 5). On this day,
the fish with the higher levels of PHB (25 g kg
1 and 50 g kg
1) the mortality of the challenged larvae that were fed with PHB was
significantly increased the activity of lipase as compared to the significantly lower than the mortality of the challenged larvae not
control group which had the lowest lipase activity. fed with PHB. At the same time, their mortality was not
significantly different from the unchallenged larvae. At 10 and
3.1.3. Fish body composition 11 days after challenge, the mortality of the challenged larvae fed
A similar average total protein content was observed for the fish with PHB was still significantly lower than the challenged larvae
of all treatments with values of 58.2  1.5%, 59.4  1.0%, 58.6  1.7% that were not fed with PHB. However, their mortality was
and 59.4  2.1% on dry weight (DW) in the control, 5 g kg
1, significantly higher than that of the unchallenged larvae.
25 g kg
1 and 50 g kg
1 PHB treatment groups, respectively
(P > 0.05). Dietary PHB supplementation significantly increased 4. Discussion
the whole-body total lipid content with 2–3% on DW when
compared to the control treatment (Table 4). Although positive effects of PHB on the growth performance of
European sea bass juveniles have been reported by De Schryver
3.1.4. Fatty acid profile et al. (2010) no significant differences in final body weight were
The fish FA profile (content and composition) following 28 days found between the different treatment groups in the current study.
of feeding on different diets is presented in Table 4. The contents of Despite the trend of a higher mean final body weight observed in

Table 4
Whole-body total lipid content (% on DW; mean  standard deviation), the content of the main groups of long chain fatty acids (C14–C24) as % on DW (mean  standard
deviation), and the composition of the main groups of long chain fatty acids (C14–C24) as % on total lipid content (in parenthesis; mean  standard deviation) in Nile tilapia
juveniles fed with different PHB experimental diets (number of replicates per treatment = 3; number of fish pooled per replicate for FAME analysis = 10).

Treatment Total lipid FA composition (n-6)/(n-3) ratio

Saturated Mono- EPA DHA Total (n-6) Total (n-3)

unsaturated
Control 25.40  0.33a 7.22  0.16a 5.99  0.16a 0.06  0.03a 1.45  0.14a 7.18  0.03a 1.91  0.13a 3.76  0.25a
(28.44  0.62a) (23.59  0.61a) (0.24  0.01a) (5.71  0.54a) (28.27  0.12a) (7.53  0.51a)

5 g kg
1 PHB 28.26  0.13b 8.23  0.35bc 6.68  0.23b 0.07  0.06a 1.65  0.02a 7.61  0.47ab 2.13  0.23a 3.58  0.16a
(29.13  1.25a) (23.64  0.80a) (0.26  0.02a) (5.83  0.08a) (26.94  1.67a) (7.54  0.80a)

25 g kg
1 PHB 27.69  1.06b 8.47  0.17c 6.79  0.25b 0.07  0.08a 1.51  0.13a 7.32  0.41ab 1.95  0.16a 3.76  0.10a
(30.58  0.60a) (24.51  0.89a) (0.26  0.03a) (5.45  0.48a) (26.44  1.48a) (7.03  0.56a)

50 g kg
1 PHB 27.92  0.13b 7.82  0.24ab 6.36  0.06ab 0.07  0.02a 1.63  0.02a 8.09  0.23b 2.10  0.03a 3.85  0.14a
(27.99  0.86a) (22.78  0.23a) (0.27  0.01a) (5.82  0.07a) (28.95  0.81a) (7.52  0.10a)

Different superscript letters within a column denote significant differences (P  0.05).

48 M.L. Situmorang et al. / Veterinary Microbiology 182 (2016) 44–49

Table 5
Effect of PHB on the cumulative mortality (%, mean  standard deviation) of gnotobiotic Nile tilapia larvae challenged with pathogenic E. ictalurigly09R via culture water and
Artemia nauplii (number of replicates per treatment = 4; initial number of fish larvae per replicate = 10). DAC = days after challenge. Different superscript letters within the
same row denote significant differences (P  0.05).

Larvae not challenged, Larvae not challenged, Larvae challenged, Larvae challenged,
not fed with PHB fed with PHB not fed with PHB fed with PHB
6 DAC 3  5a 3  5a 3  5a 3  5a
7 DAC 3  5a 3  5a 10  8a 3  5a
8 DAC 8  5a 3  5a 13  13a 5  6a
9 DAC 10  0a 3  5a 30  18b 10  8a
10 DAC 20  12a 18  10a 60  8c 40  12b
11 DAC 28  10a 20  8a 70  8c 50  8b

the PHB treatment groups (258–284 mg) when compared to the The increased lipid deposition can be undesirable from a
control group (218 mg) it can thus only be concluded that PHB did commercial perspective because it would mean that the higher
not have negative effects on the growth of Nile tilapia juveniles. weight of the fish in the PHB treatments (although not significant)
The significantly higher SGR in the 5 g kg
1 PHB treatment can results from extra lipid and not extra muscle or “real growth”. On
likely be explained by the slightly higher mortality due to the other hand, the increase in total lipid content can be favorable
aggressive behavior resulting in a higher contribution of stron- for human consumption to a certain degree, as lipids play an
ger/larger fish in the calculation of the final average body weight essential role as energy and FAs source for humans, both for
for this treatment. The observations on the effect of PHB on the immediate utilization by the body and in laying down a storage
growth of the fish correspond with the study by Najdegerami et al. depot (adipose tissue) for later utilization when food intake is
(2012) where, despite the observation of higher final body weight reduced. They can also act as a vehicle for the ingestion and
in PHB treatments, no significant effect of PHB on the growth absorption of fat-soluble vitamins (Medeiros and Wildman, 2013).
performance of Siberian sturgeon fingerlings could be concluded. Fats as a source of essential FAs (omega-3 and omega-6) are
The use of PHB to significantly promote fish growth thus seems to specifically of interest as they for example serve as specific
be species specific. Possibly, the fact that sea bass is carnivorous precursors for eicosanoids that regulate numerous cell and organ
while sturgeon and tilapia are omnivorous plays a role. Fish of functions in humans (FAO, 2010; Uauy et al., 2000). It was thus
different trophic levels indeed differ in anatomy and functionality interesting to verify if the increment of total lipid resulting from
of the digestive tract which may influence the metabolization of PHB supplementation corresponded to an increase in essential FAs
feed components (Smith, 1980). In addition to species, the age or in the fish or that more neutral (or non-essential) fat which does
developmental stage of the fish will also be of importance in the not contribute to the quality of the tilapia as a consumption
PHB effect as it was most recently found that PHB did induce product was accumulated.
significant growth promoting effects in tilapia fry (unpublished Earlier, it was reported by Najdegerami et al. (2012) that PHB
data). For Siberian sturgeon larvae, then again, PHB seemed to significantly increased the linoleic acid (LA) and total (n-6) FAs
negatively affect the growth performance (Najdegerami et al., content of the liver of Siberian sturgeon fingerling fed a 5 g kg
1
2015). Further research focussing specifically on the digestibility of PHB diet. In another study, PHB was found to increase the total
PHB in fish of different trophic level and/or age could provide more monounsaturated and total (n-3) FAs content of giant tiger prawn
information on this aspect. postlarvae (Ludevese-Pascual, personal communication). Similar-
Digestive enzyme activities can be used as indicators of ly, an increasing trend was observed in the total saturated, total
digestive processes and fish nutritional conditions (Lazo et al., monounsaturated FAs and total (n-6) FAs content (% on DW) for the
2000; Ueberschar, 1988). It has been reported that manipulation of Nile tilapia juveniles fed with PHB in this study. However, PHB did
diets causes immediate changes in activities of digestive enzymes not affect the composition of FAs groups when expressed as % on
(Mohapatra et al., 2012). This specifically holds true for fishes with total lipid content. This indicates that the fish FA profile was not
relatively broad diets (German et al., 2004). In this study, dietary affected by PHB supplementation. The FAME analysis procedure
PHB supplementation resulted in a significantly higher lipase performed in this study only measured the long chain fatty acids
activity in the 25 and 50 g kg
1 PHB-fed fish group, while a similar (C14–C24) and did not provide information on the composition of
activity of trypsin, amylase, and pepsin was observed among all SCFAs (C2–C6) or medium chain fatty acids (C8–C12). As the
treatments. Lipases are the enzymes responsible for the break- degradation of dietary PHB is hypothesized to results in the release
down of dietary lipid complexes within the intestinal lumen of SCFAs, likely b-HB (De Schryver et al., 2010; Defoirdt et al.,
(Karasov and Hume, 1997). The 20–40% increase in lipase activity in 2009), it will be interesting to evaluate the effect of PHB on the fish
the 25 and 50 g kg
1 PHB treatment groups suggests that the SCFAs profile in future research.
dietary PHB stimulated the digestion of dietary lipids, providing The gnotobiotic challenge test was used to investigate if PHB
more fatty acids available for absorption and esterification into has a similar disease protecting effect for fish as it does for
complex lipids. Such increase has earlier been shown to be crustaceans. In this study, E. ictaluri gly09R, a pathogen known to
physiologically relevant as a study by Essa et al. (2010) reported a cause mortality in tilapia culture, was used (Soto et al., 2012). The
higher growth and feed utilization resulting from a 35–80% experiment showed that PHB supplementation to gnotobiotic Nile
increase in gut lipase activity in Nile tilapia fingerlings (25 g). The tilapia larvae provided significant protection against the challenge,
higher lipase activity may thus have contributed to the increase in although the protection was not complete in comparison to a
total lipid content as observed in the fish from the PHB treatment negative control. These observations are similar to the ones of Thai
groups. Higher lipid deposition, however, does not equate to faster et al. (2014) in freshwater prawn and Laranja et al. (2014) in giant
growth and may indicate an inefficient use of nutrients (Hixson, tiger prawn. While it has been shown that the use of PHB limits the
2014). Indeed, Hanley (1991) indicated that tilapia that were able pathogenicity and the presence of presumptive vibrios in shrimp
to store significant quantities of lipid in their carcass and viscera (Defoirdt et al., 2007; Thai et al., 2014), it remains to be determined
could not utilize this energy source to improve growth. if PHB also worked through affecting the growth or activity of E.
 M.L. Situmorang et al. / Veterinary Microbiology 182 (2016) 44–49 49

ictalurigly09R in the tilapia or that it may have acted as an German, D.P., Horn, M.H., Gawlika, A., 2004. Digestive enzyme activities in
immunostimulant as described by Baruah et al. (2015) for brine herbivorous and carnivorous Prickelback fishes (Teleostei: Stichaeidae):
ontogenetic, dietary and phylogenetic effects. Physiol. Biochemi. Zool. 77 (5),
shrimp. The availability of the gnotobiotic Nile tilapia challenge 789–804.
test creates the opportunity for a detailed investigation of the Hanley, F., 1991. Effects of feeding supplementary diets containing varying levels of
effects of PHB exposure on pathogen growth and virulence under lipid on growth, food conversion and body composition of Nile tilapia
Oreochromis niloticus (L.). Aquaculture 93, 323–334.
microbiologically controlled conditions. Hixson, S., 2014. Fish nutrition and current issues in aquaculture: the balance in
providing safe and nutritious seafood, in an environmentally sustainable
5. Conclusion and perspectives manner. J. Aquac. Res. Development. 5, 234–243.
Holm, H., Krogdahl, A., Hanssen, L.E., 1988. High and low inhibitor soybean meals
affect human duodenal proteinase activity differently: in vitro comparison of
In general, the findings of this study suggest that PHB does not proteinase inhibition. J. Nutr. 118, 521–525.
have a negative effect on the growth of Nile tilapia juveniles. A Iijima, N., Tanaka, S., Ota, Y., 1998. Purification and characterization of bile salt-
activated lipase from the hepatopancreas of red sea bream Pagrus major. Fish
trend of increasing body weight was observed in all PHB treatment
Physiol. Biochem. 18, 59–69.
groups, however, the increase was not statistically significant Karasov, W., Hume, I., 1997. The vertebrate gastrointestinal system. In: Dantzler, W.
compared to the control group. Furthermore, lipid digestion and (Ed.), Handbook of Physiology. Oxford University Press, New York, pp. 407–480
deposition seemed to be increased by the dietary PHB supplemen- Section 13: Comparative Physiology.
Laranja, J.L.Q., Ludevese-Pascual, G.L., Amar, E.C., Sorgeloos, P., Bossier, P., De
tation. We hypothesize that the altered lipid utilization may have Schryver, P., 2014. Poly-b-hydroxybutyrate (PHB) accumulating Bacillus spp.
resulted from diet-induced alterations in the lipase activity. A improve the survival, growth and robustness of Penaeus monodon (Fabricius,
challenge test making use of a gnotobiotic Nile tilapia larvae 1798) postlarvae. Vet. Microbiol. 173, 310–317.
Lazo, J.P., Holt, G.J., Arnold, C.R., 2000. Ontogeny of pancreatic enzymes in larval red
system confirmed that PHB is also effective as an antipathogenic drum Sciaenops ocellatus. Aquacult. Nutr. 6, 183–192.
strategy for fish. Further research needs to focus on the effect of Lepage, G., Roy, C.C., 1984. Improved recovery of fatty acid through direct
PHB on the modulation of the digestive functionality in fish of transesterification without prior extraction or purification. J. Lipid Res. 25,
1391–1396.
different trophic levels and on determining the way in which PHB Marques, A., Dhont, J., Sorgeloos, P., Bossier, P., 2004. Evaluation of different yeast
provides protection against pathogens in fish. cell wall mutants and microalgae strains as feed for gnotobiotically grown brine
shrimp Artemia franciscana. J. Exp. Mar. Bio. Ecol. 312, 115–136.
Medeiros, D.M., Wildman, R.E.C., 2013. Advance Human Nutrition, 3rd ed. Jones &
Acknowledgements
Barlett Learning, Burlington, USA, pp. 391.
Métais, P., Bieth, J., 1968. Determination de l’amylase par une microtechnique.
This work was performed under financial support from a Annales de Biologie Clinique (Paris) 26, 133–142.
Mohapatra, S., Chakraborty, T., Prusty, A.K., Das, P., Paniprasad, K., Mohanta, K.N.,
Doctoral Grant from The World Bank, USA, to the first author under
2012. Use of different microbials probiotics in the diet of rohu, Labeo rohita
the Japan Indonesia Presidential Scholarship (JIPS) Program. Peter fingerlings: effects on growth, nutrient digestibility and retention, digestive
De Schryver is supported as a postdoctoral fellow by the Research enzyme activities and intestinal microflora. Aquacult. Nutr. 18, 1–11.
Foundation—Flanders (FWO), Belgium. The authors thank the Najdegerami, E.H., Tran, T.N., Defoirdt, T., Marzorati, M., Sorgeloos, P., Boon, N.,
Bossier, P., 2012. Effects of poly-beta-hydroxybutyrate (PHB) on Siberian
Laboratory of Aquaculture & Artemia Reference Center (ARC) staff sturgeon (Acipenser baerii) fingerlings performance and its gastrointestinal tract
for the excellent technical support. microbial community. FEMS Microbiol. Ecol. 79, 25–33.
Najdegerami, E.H., Baruah, K., Shiri, A., Rekecki, A., Van den Broeck, W., Sorgeloos, P.,
Boon, N., Bossier, P., De Schryver, P., 2015. Siberian sturgeon (Acipenser baerii)
References larvae fed Artemia nauplii enriched with poly-b-hydroxybutyrate (PHB): effect
on growth performance, body composition, digestive enzymes, gut microbial
AOAC, 2000. in: Horwitz, W. (Ed.), Official Methods of Analysis of AOAC community, gut histology and stress tests. Aquac. Res. 46, 801–812.
International. AOAC, Arlington, VA, USA. Ng, W.K., Romano, N., 2013. A review of the nutrition and feeding management of
Anderson, A.J., Dawes, E.A., 1990. Occurrence metabolism, metabolic role, and farmed tilapia throughout the culture cycle. Rev. Aquacult. 5, 220–254.
industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54, 450–472. Ng, W.K., Chong, C.Y., 2004. An overview of lipid nutrition with emphasis on
Balarin, J.D., Halfer, R.D., 1982. The intensive culture of tilapia in tanks, raceways and alternative lipid sources in tilapia feeds. In: Bolivar, R.B., Mair, G.C.,
cages. In: Muir, J.F., Roberts, R.J. (Eds.), Recent Advances in Aquaculture. Crom Fitzsimmons, K. (Eds.), New Dimensions in Farmed Tilapia. Sixth International
Helm, London, pp. 265–356. Symposium on Tilapia in Aquaculture. American Tilapia Association, USA, pp.
Baruah, K., Huy, T.T., Norouzitallab, P., Niu, Y., Gupta, S.K., De Schryver, P., Bossier, P., 241–248.
2015. Probing the protective mechanism of poly-ß-hydroxybutyrate against Nhan, D.T., Wille, M., De Schryver, P., Defoirdt, T., Bossier, P., Sorgeloos, P., 2010. The
vibriosis by using gnotobiotic Artemia franciscana and Vibrio campbellii as host- effect of poly b-hydroxybutyrate on larviculture of the giant freshwater prawn
pathogen model. Sci. Rep. 5, 9427. Macrobrachium rosenbergii. Aquaculture 302, 76–81.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of Situmorang, M.L., Dierckens, K., Mlingi, F.T., Van Delsen, B., Bossier, P., 2014.
microgram quantities of protein utilizing the principle of protein-dye binding. Development of a bacterial challenge test for gnotobiotic Nile tilapia
Anal. Biochem. 72, 248–254. Oreochromis niloticus larvae. Dis. Aquat. Organ. 109, 23–33.
De Schryver, P., Sinha, A., Kunwar, P., Baruah, K., Verstraete, W., Boon, N., De Boeck, Smith, L.S., 1980. Digestion in teleost fishes. Fish Feed Technology—ADCP/REP/80/11.
G., Bossier, P., 2010. Poly-ß-hydroxybutyrate (PHB) increases growth Food and Agriculture Organisation of the United Nations, Rome, pp. 395.
performance and intestinal bacterial range-weighted richness in juvenile Soto, E., Maziel, A., Riofrio, A., Martinez, A., Cabrejos, M.E., 2012. Edwardsiella ictaluri
European sea bass, Dicentrarchus labrax. Appl. Microbiol. Biotechnol. 86, 1535– as the causative agent of mortality in cultured Nile tilapia. J. Aquat. Anim. Health
1541. 24, 81–90.
Defoirdt, T., Halet, D., Vervaeren, H., Boon, N., De Wiele, T., Sorgeloos, P., Bossier, P., Sui, L., Cai, J., Sun, H., Wille, M., Bossier, P., 2012. Effect of poly-beta-hydroxybutyrate
Verstraete, W., 2007. The bacterial storage compound poly-ß-hydroxybutyrate on Chinese mitten crab, Eriocheir sinensis, larvae challenged with pathogenic
protects Artemia franciscana from pathogenic Vibrio campbellii. Environ. Vibrio anguillarum. J. Fish Dis. 35, 359–364.
Microbiol. 9, 445–452. Thai, T.Q., Wille, M., Garcia-Gonzalez, L., Sorgeloos, P., Bossier, P., De Schryver, P.,
Defoirdt, T., Boon, N., Sorgeloos, P., Verstraete, W., Bossier, P., 2009. Short-chain fatty 2014. Poly-ß-hydroxybutyrate content and dose of the bacterial carrier for
acids and poly-b-hydroxyalkanoates: (new) biocontrol agents for a sustainable Artemia enrichment determine the performance of giant freshwater prawn
animal production. Biotechnol. Adv. 27, 680–685. larvae. Appl. Microbiol. Biotechnol. 98, 5205–5215.
Defoirdt, T., Sorgeloos, P., Bossier, P., 2011. Alternatives to antibiotics for the control Tokiwa, Y., Calabia, B.P., 2007. Biodegradability and biodegradation of polyesters. J.
of bacterial disease in aquaculture. Curr. Opin. Microbiol. 14, 251–258. Polym. Environ. 15, 259–267.
Doyle, C., Tanner, E., Bonfield, W., 1991. In vitro and in vivo evaluation of poly Uauy, R., Mena, P., Rojas, C., 2000. Essential fatty acids in early life: structural and
hydroxybutyrate and of poly hydroxybutyrate reinforced with hydroxyl apatite. functional role. Proc. Nutr. Soc. 59, 3–15.
Biomaterials. 12, 841–847. Ueberschar, B., 1988. Determination of the nutritional condition of individual
Essa, M., El Serafy, S., El Ezabi, M., Daboor, S., Esmael, N., Lall, S., 2010. Effect of marine fish larvae by analyzing their proteolytic enzyme activities with a highly
different dietary probiotics on growth, feed utilization and digestive enzymes sensitive technique. Meeresforschung 32, 144–154.
activities of Nile tilapia, Oreochromis niloticus. J. Arab. Aquat. Soc. 5, 143–162. Ways, P., Hanahan, D.J., 1964. Characterization and quantification of red cell lipids in
FAO, 2010. Fats and fatty acids in human nutrition: Report of an expert consultation. normal man. J. Lipid Res. 5, 318–328.
Food and Agriculture Organization of the United Nations, Rome. Zambonino-Infante, J.L., Cahu, C.L., 1994. Early weaning of sea bass (Dicentrarchus
Folch, J., Lees, M., Sloane-Stanley, G., 1957. A simple method for the isolation and labrax) larvae with a compound diet: effect on digestive enzymes. Comp.
purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Biochem. Physiol. 109A, 212–222.