Dietary Probiotic Pediococcus acidilactici MA18/5M Improves
the Growth, Feed Performance and Antioxidant Status of
Penaeid Shrimp Litopenaeus stylirostris:
A Growth-Ration-Size Approach
Mathieu Castex 1, *, Eric Leclercq 1, Pierrette Lemaire 2and Liêt Chim 2,3
Citation: Castex, M.; Leclercq, E.;
Lemaire, P.; Chim, L. Dietary
Probiotic Pediococcus acidilactici
MA18/5M Improves the Growth,
Feed Performance and Antioxidant
Status of Penaeid Shrimp Litopenaeus
stylirostris: A Growth-Ration-Size
Approach. Animals 2021,11, 3451.
Academic Editors: Cedric J. Simon,
Mauricio G.C. Emerenciano,
Artur Rombenso, Felipe do
Nascimento Vieira and Ravi Fotedar
Received: 18 September 2021
Accepted: 30 November 2021
Published: 3 December 2021
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1LALLEMAND SAS, 19 rue des Briquetiers, 31702 Blagnac, France; email@example.com
2IFREMER, UnitéLagons, Ecosystèmes et Aquaculture Durable en Nouvelle Calédonie (LEAD), B.P. 2059,
98846 Nouméa, New Caledonia, France; firstname.lastname@example.org (P.L.); email@example.com (L.C.)
3IFREMER, Laboratoire BRM/PBA, Rue de l’Ile d’Yeu, 44311 Nantes, France
*Correspondence: firstname.lastname@example.org; Tel.: +33-620871370
Probiotics are increasingly documented to confer health and performance beneﬁts
across farmed animals. The study assessed the effect of dietary supplementation with the single-
strain probiotic Pedicococcus acidilactici MA18/5M on the growth, nutritional indices, and metabolic
status of the adult western blue shrimp, Litopenaeus stylirostris. The aim was to estimate its potential
at optimizing the performance of the penaeid feed and shrimp farming industry. Supplementation
with P. acidilactici MA18/5M improved the feed conversion efﬁciency and daily growth rate across
ﬁxed ration sizes; and decreased both the maintenance and optimal ration size for growth. This
appeared linked to a better use of dietary carbohydrates as shown by a higher
free-glucose and glycogen concentration in the digestive gland. Interestingly, P. acidilactici intake was
also associated with a higher antioxidant status which may be linked to enhanced carbohydrates
utilization. Using a ﬁxed ration size approach under controlled laboratory conditions, the study
documented a clear potential for P. acidilactici MA18/5M to enhance the growth, feed efﬁciency
and metabolic health of adult penaeid shrimp during on-growing. These ﬁndings raise interesting
prospects to optimize penaeid feed formulation and the performance of the shrimp-farming industry.
Probiotics are increasingly documented to confer health and performance beneﬁts across
farmed animals. The aim of this study was to assess the effect of a constant daily intake of the single-
strain probiotic Pedicococcus acidilactici MA18/5M (4 ×108CFU.day−1.kg−1shrimp) fed over ﬁxed,
restricted ration sizes (1% to 6% biomass.day
) on the nutritional performance and metabolism of
adult penaeid shrimp Litopenaeus stylirostris (initial body-weight, BWi = 10.9 ±1.8 g). The probiotic
signiﬁcantly increased the relative daily growth rate (RGR) across all ration size s tested (Mean-RGR
0.08 and 0.61
for the control and probiotic groups, respectively) and
decreased the maintenance ration (Rm) and the optimal ration (Ropt) by 18.6% and 11.3%, respec-
tively. Accordingly, the probiotic group exhibited a signiﬁcantly higher gross (K1) and net (K2) feed
conversion efﬁciency with average improvement of 35% and 30%, respectively. Enhanced nutritional
performances in shrimps that were fed the probiotic P. acidilactici was associated with, in particular,
-amylase speciﬁc activity (+24.8
5.5% across ration sizes) and a concentration
of free-glucose and glycogen in the digestive gland at ﬁxed ration sizes of 3% and below. This
suggests that the probiotic effect might reside in a better use of dietary carbohydrates. Interestingly, P.
acidilactici intake was also associated with a statistically enhanced total antioxidant status of the diges-
tive gland and haemolymph (+24.4
7.8% and +21.9
). As supported
by knowledge in other species, enhanced carbohydrate utilization as a result of
may fuel the pentose-phosphate pathway, generating NADPH or directly enhancing OH-radicals
scavenging by free glucose, in turn resulting in a decreased level of cellular oxidative stress. In
conclusion, the growth-ration method documented a clear contribution of P. acidilactici MA18/5M
on growth and feed efﬁciency of on-growing L. stylirostris that were fed ﬁxed restricted rations
Animals 2021,11, 3451. https://doi.org/10.3390/ani11123451 https://www.mdpi.com/journal/animals
Animals 2021,11, 3451 2 of 21
under ideal laboratory conditions. The effect of the probiotic on
-amylase activity and carbohydrate
metabolism and its link to the shrimp’s antioxidant status raises interesting prospects to optimize
dietary formulations and helping to sustain the biological and economic efﬁciency of the penaeid
Keywords: probiotic; Pediococcus acidilactici; shrimp; growth; carbohydrate; antioxidant status
Aquaculture widely contributes to the availability of aquatic food for human consumption
and relies on high-quality feed to ensure the health and performance of the animals and the
sustainability of its industry. Research into the use of probiotics for aquatic animals has increased
with the demand for environmentally friendly and sustainable aquaculture [
were originally defined as microbial dietary supplements which bring beneficial effects to the
] and are commonly viewed as prophylactic supplements in human health [
] detail the various developments made in the application of probiotics in aquatic
species, including shrimp. In aquaculture, probiotics are usually used as biocontrol agents for
preventing disease and/or increasing resistance to pathogens [
] and several possible action
mechanisms have been suggested. Among these, the competitive exclusion of pathogenic
] and enhancement of the immune and antioxidant defense systems against
pathogenic micro-organisms [
] have been widely invoked. For instance, the probiotic
strain Pediococcus acidilactici MA18/5M has been the subject of a number of evaluations with
regard to the health of the penaeid shrimp Litopenaeus stylirostris, and its dietary use was shown
to be beneficial against vibriosis [17,18,22].
Probiotics are also often used as growth promoting agents in farmed animals [
and as a digestibility enhancer in ruminants [
]. Several studies have recently reported
that probiotic bacteria are good candidates for improving nutrient digestion and the growth
of aquatic organisms [
]. Beneﬁts to the host have been reported to include an improved
feed utilisation and feed value by supplying beneﬁcial dietary compounds (vitamin B12,
biotin, carotenoids, amino acids), by detoxifying potentially harmful compounds in feeds
and/or by an enzymatic contribution to digestion [
]. For example, Ref. [
that abalone (Haliotis midae) that were fed a kelp diet supplemented with Pseudoalteromonas
sp. strain C4 exhibited an increased growth rate compared to abalone fed standard kelp.
They suggested that the probiotic can play an important role in the nutrition of farmed
abalone in three ways: (i) pre-digestion of alginate in kelp-based feed, (ii) increased
alginate lyase activity in the abalone digestive tract and (iii) utilisation of strain C4 as a
protein source. Moreover, probiotics are also considered to inﬂuence digestive processes by
enhancing the population of beneﬁcial micro-organisms [
], microbial enzyme activity
and the intestinal microbial balance [
]. However, to date, probiotic studies carried out
with shrimps have mainly focused on their increased resistance to disease [
population growth performance [19,32–35].
The aim of the present study was to specify the effect of P. acidilactici on the growth
and nutrition of the shrimp L. stylirostris. To this end, we determined the ration size
for maintenance and optimal growth by means of the growth-ration method initially
proposed for ﬁsh by [
] and largely applied in ﬁsh. Its main function is to determine the
quantitative daily nutritional and energy requirements for maintenance and optimal growth
as well as the impact of environmental factors [
]. Surprisingly, this fundamental
GR-method remains seldom applied and quantitative requirements are comparatively
infrequently determined for penaeid shrimps. Refs. [
] based their studies on the
GR-relation to determine the effect of feeding frequency and natural productivity of the
pond on growth and feed efﬁciency of Penaeus merguiensis and L. stylirostris. Ref. [
used the same approach to estimate the daily quantitative protein requirements of juvenile
Litopenaeus vannamei. However, most studies have evaluated the effect of qualitative
Animals 2021,11, 3451 3 of 21
variations of the feed nutritional proﬁle on growth and feed conversion. Indeed, as
mentioned by [
], research on the protein requirements of penaeid shrimp has largely been
concerned with evaluations of optimal dietary protein level and not with the quantitative
protein requirement, since ad libitum feeding is generally used. The use of the GR-relation
and the quantiﬁcation of the daily requirements obtained from it could provide useful
information to maximize penaeid shrimp production [
]. Moreover, this method could be
used to evaluate and optimize the use of dietary additives.
Importantly, in many studies on crustaceans, the rearing tank is generally used as
the experimental unit and a simple analysis of variance is applied to the tank’s mean
data. However, Ref. [
] highlighted the advantage of using individual measurements and
nested designs in aquaculture experiments. In order to analyse such designs without pseu-
], a nested analysis of variance has been identiﬁed as the correct method to
]. Nested ANOVA maintains both between-tank and within-tank variability in the
analysis, and therefore reduces the risk of drawing invalid conclusions as can occur when
using a simple analysis of variance [
]. New techniques and methods are now available
for measuring several variables at the individual level for ﬁsh, as mentioned by [
individual tagging of the animals is one such method [
]; however, its use for individual
shrimp growth measurements remains undocumented.
In this study, we combined the GR-method and individual tagging of the test speci-
mens in order to precisely measure the key nutritional parameters (maintenance and the
optimal rations, scope for growth, gross and net conversion efﬁciency) and accurately
assess the putative effects of the probiotic on the growth and feed efﬁciency. Furthermore,
a biochemical analysis of free glucose, glycogen, digestive enzyme activities and total
antioxidant status (TAS) in the haemolymph and digestive gland was carried out to gain
further insight into the probiotic effect at physiological level. To the authors knowledge, no
such approach has been previously applied to assess the effect of probiotic supplementation
on nutritional parameters in shrimps.
2. Materials and Methods
The trial was conducted at the Saint Vincent Aquaculture Research Station of IFREMER
located on the west coast of New Caledonia (Latitude 21◦470S, Longitude 165◦450E).
2.1. Experimental Diets and Dietary Probiotic
The basal diet was formulated (Table 1; 43.8% protein, 10.0% lipid; 2.0% ﬁber, 4502
gross energy) and processed internally as follows: The ingredients were grinded
using a laboratory grinder with a 1 mm screen and the meal was mixed with oil and water
(30%) using a horizontal mixer. The mixture was then extruded using a meat grinder
through a 3 mm die, the feed strands were dried (60
C, 24 h) in a drying-oven to a residual
moisture of below 10% and then broken-up into pellets from 4 to 5 mm in length.
The commercial probiotic tested in this study (Bactocell PA 10; Lallemand S.A.S,
Blagnac, France) consisted of live, freeze-dried Pediococcus acidilactici MA 18/5M (Institut
Pasteur, Paris, France) formulated in a powder form to a concentration of 10
of product. The probiotic was top-coated on the pellets using 3% of ﬁsh oil as a carrier.
Probiotic concentrations in each diet were reviewed after feed production against the
target concentrations (Table 2) through homogenisation of the feed in peptone water, serial
dilution, plating of the selected dilutions on the lactobacilli-speciﬁc de Man, Rogosa &
Sharpe (MRS) plates and counting the number of Colony Forming Units after incubation
C; 48 h). The control diet was also top-coated with 3% ﬁsh oil and checked for possible
contamination by the probiotic strain. All feeds were stored in sealed 5-litre boxes at room
temperature (~20 ◦C) until use.
Animals 2021,11, 3451 4 of 21
) Raw material composition, (
) proximate composition and (
) energy content of the basal
(a) Ingredients (g/kg)
LT ﬁsh meal (a) 300
Soybean meal (b) 200
Wheat meal (c) 370
Wheat gluten 70
Fish oil 20
Soy oil 20
Soy lecithin (d) 20
Shrimp Vitamin premix (e) 0.5
Shrimp trace mineral premix (f) 1
Stay C (g) 0.4
(b) Proximate Analysis
Protein (1) (%, DM basis) 43.8
Fat (2) (%, DM basis) 10
Fiber (3) (%, DM basis) 2
Ash (4) (%, DM basis) 6.9
(c) Energy Content (kcal.kg−1)
Gross energy (5) 4502
Digestible energy (6) 3376
LT, low temperature; DM, dry matter.
Chilean low temperature ﬁsh meal from anchovy and jack mackerel;
Dehulled soybean meal, solvent extracted;
Whole wheat gran for animal feed;
lecithin, Decatur, IL, USA);
SICA Cie (Noumea, New Caledonia, France);
Vitamin C (330 mg.kg
DSM, Basel, Switzerland) ISO5983 standard;
NF V18-117/B standard;
NF V18-101 standard;
Determined by calorimetric bomb (Parr
, USA, calibrated by benzoic acid).
(6) Calculated using the concentration of chromic oxide in feed and feces (Not presented).
Expected and measured probiotic concentration (Pediococcus acidilactici) in the probiotic diet
prepared at different concentration for each ration size in order to achieve a daily probiotic intake of
SD. Measured count was always within the acceptable range
(<0.5 log difference between expected and measured count).
Daily Ration Size (% BMi.day−1)P. acidilactici Count (×107CFU.g−1Feed)
1 4.00 3.8 ±0.4
2 2.00 2.5 ±0.2
3 1.33 1.5 ±0.3
4 1.00 0.87 ±0.05
6 0.67 0.65 ±0.05
BMi, initial tank biomass.
2.2. Animals, Tagging and System
Locally sourced L. stylirostris (20-day post-larvae, PL20) were stocked at a density of
20 post-larvae per m
within a single earthen pond (1000 m
), and reared under standard
semi-intensive practices in New Caledonia for 4-month until they reached the desired size
for the trial. In brief, shrimp were fed twice daily with a commercial formulated feed; the
feeding rate was adjusted weekly according to the estimated mean body-weight (BW) and
the amount of remaining feed in the feeding trays was assessed two hours after feeding.
Experimental shrimps were caught from an earthen pond using a cast net, transported
in 50 L plastic containers ﬁlled with seawater, randomly stocked into the experimental
tanks (6 shrimps/tank) and were permitted to acclimate for 1-week to the experimental con-
ditions. Following acclimation, shrimps were individually colour tagged (NMT Elastomer
System, Norwest Marine Technology, Shaw Island, WA, USA) by sub-cuticle injection in
the last segment of the abdomen, just above the telson. Within each tank, it was possible to
distinguish each specimen using 5 distinct tag colours and one specimen was left untagged
Animals 2021,11, 3451 5 of 21
but handled and sham-injected accordingly. Following tagging and BW measurement,
animals were returned to their original tanks, at which point the trial started.
The trial was carried out in 30 self-cleaning circular polyester tanks (0.92 m
surface area; 536 L capacity) that were continuously supplied with natural seawater (100%
renewal daily) pumped-ashore the adjacent lagoon, sand-ﬁltered and stored in an elevated
earthen reservoir for gravity supply. The water renewal rate was set at 400%.day
and aeration was provided to each tank. The temperature was measured continuously
using an automatic recording probe (Optic StowAway
Temp; Onset, MA, USA). Water
quality parameters over the trial’s duration were DO > 80%; temperature 27.2
salinity = 35.0 ±0.1 ppt.
2.3. Experimental Design, Feeding and Sampling
The 27-day experiment was conducted using 180 sub-adult L. stylirostris at an initial
BW (BWi) of 10.93
1.78 g. The experiment tested two diet conditions (probiotic vs.
control) with ﬁve ﬁxed ration sizes (1%, 2%, 3%, 4% and 6% BMi.day
; where BMi is
initial biomass) per diet with three of the tanks randomly assigned to each treatment (30
tanks, 6 shrimp.tank
). The set-feed rations were determined by prior experimentation,
which had shown that they were completely consumed under our experimental conditions,
with apparent satiation circa 7% BM.day
. The probiotic treatment targeted, based on
prior knowledge [
], a daily P. acidilactici 18/5M intake of 4
across ration sizes such that the probiotic incorporation within each diet group was adjusted
to the ﬁve pre-determined ration size (Table 2). The daily pre-weighed ration per tank was
delivered in four equally sized meals distributed at 7.00 a.m., 1.00 p.m., 7.00 p.m. and 1.00
a.m. using automatic feeders.
Individually tagged specimen were each measured for BW (
0.01 g) after carefully
drying on soft paper at the start (immediately after colour tagging) and at the end of the
trial (on the 28th day at 8.00 a.m., seven hours after the last feeding). At the end of the trial,
four shrimps per tank were further sacriﬁced and immediately sampled for haemolymph
and the digestive gland. Only shrimps in intermoult (stage C-D0) were used as digestive
enzyme activity and other physiological parameters change during the moulting stage. To
do so, 200
L haemolymph was withdrawn from the ventral sinus cavity using a 1 mL
sterile syringe, ﬁtted with a 23-gauge needle. Haemolymph samples were immediately
diluted in a pre-cooled saline-sodium citrate buffer (SCC; 30 mM trisodium citrate, 0.34
M sodium chloride, 1 mM EDTA) and snap-frozen in liquid nitrogen prior to storage at
C until analysis. Digestive glands were removed, snap-frozen in liquid nitrogen and
stored at −80 ◦C until analysis.
2.4. Analytical Protocols
2.4.1. Samples Preparation
The diluted haemolymph samples were thawed under refrigeration, vortexed and
assayed for glucose and Total Antioxidant Status (TAS). The digestive glands were thawed
under refrigeration, divided into two parts, each of which was weighed. One part was
homogenised using an ultra-turrax
in a 10 mM Tris buffer (1 mM DTPA, 1 mM PMSF, pH
7.4) for protein, glucose, glycogen,
-amylase and trypsin activity assays and the other part
was homogenised in an SCC buffer for TAS determination. Prior to analysis, the digestive
gland homogenates were centrifuged (4000 rpm, 10 min, 4
C) and the supernatant from
two shrimps from the same tank were pooled iso-volumetrically.
2.4.2. Biochemical Analysis
Total soluble proteins were determined in accordance with [
] with bovine serum
albumin (BSA) standard. Glucose levels were determined using a commercial kit (Glucose
RTU; bioMérieux, Craponne, France) based on the enzymatic conversion of glucose into
quinoneimine and its colorimetric quantiﬁcation at 505 nm. The assay was adapted to
microplate manipulations following the manufacturer’s recommendation. Glycogen was
Animals 2021,11, 3451 6 of 21
extracted in the presence of sulphuric acid and phenol [
] as follows: Samples were
ﬁrst homogenised in trichloro-acetic acid (TCA 5%; 2 min, 16,000 rpm) then centrifuged
(5 min, 3000 rpm). This procedure was carried out twice, the supernatants were then
pooled, vortexed and 500
L of supernatant was pipetted into a tube and mixed with
ﬁve volumes of 95% ethanol. The tubes were then left to precipitate in an oven (37
h) and centrifuged (3000 rpm, 15 min). The glycogen pellet was dissolved through the
addition of boiling water and concentrated sulphuric acid and phenol. The extract was
then transferred into a microplate reader (four replicate per sample) and read at 490 nm.
-amylase activity was assayed by the Bernfeld’s method [
] using 1% soluble
starch in phosphate buffer (20 mM; pH 7) as the substrate, 37
C incubation, and an indirect
measurement of the maltose released by DNS (3,5-dinitrosalicylic acid) colorimetry at 570
nm. One unit of enzymatic activity is deﬁned as 1 mg of maltose liberated per min at 37
and expressed as total activity (U.mg
). Trypsin was assayed by its amidase activity
using benzoyl-Arginine-p-nitroanalide (BAPNA) as the substrate, following the method
]. Assays were initiated by the addition of sample supernatant, and the release
of p-nitroanalide was measured at 410 nm over 15 min. A positive control of 3 mg.mL
trypsin (SIGMA) was used. One activity unit was expressed as 1
mol of p-nitroanilide
Total Antioxidant Status (TAS) was determined using a commercial colorimetric kit
(Randox TAS Assay; Randox Co., Antrim, UK). The tests quantify the total amount of an-
tioxidants in blood by inhibiting the transformation of 2,2-azino-di-[3-ethylbenzthiazoline
) into the radical cation (ABTS
) in the presence of a peroxidase
(metmyoglobin) and H2O2with an absorbance reading at 600 nm.
2.5. Calculations: Growth and Nutritional Parameters
2.5.1. Relative Daily Growth Rate (RGR), K1 and K2
The relative daily growth rate (RGR) was expressed as a percentage of BWi and
calculated as RGR
BWi)), where BWi and BWf are the initial
and ﬁnal body-weight, respectively, and d is the number of days between measurements.
For each modality (treatment and ration size), RGR was ﬁrst determined at an individual
level (tagged specimens) and then at tank levels based on individual’s RGR, thereby
removing any mortality from the dataset and providing a more robust estimation of RGR.
The gross feed conversion efﬁciency (K1) expresses the capacity to convert feed into
body tissues. The net feed conversion efﬁciency (K2) provides a measure of the capacity
to convert the amount of food available for growth, which is equal to the amount of feed
consumed in excess of the maintenance ration (Rm). Both K1 and K2 data were determined
for each tank according to [
] as K1 = (RGR/R)
100 and K2 = (RGR/(R
where RGR is the mean relative daily growth rate per tank, R the ration ingested per tank
and Rm is the maintenance ration per tank, all expressed in (% BM).
2.5.2. Growth-Ration (GR) and K1-Ration (KR) Curve Models
The experimental design did not allow for quantifying the shrimps’ individual feed
consumption. Thus, we cannot obtain nutritional parameters at the individual level.
However, the overall consumption of the ration provides a measurement of the feed
consumption of the shrimps at the tank level. Then, in order to determine the correct
parameters to follow, we considered a growth–ration model according to RGR means per
tank. For each dietary treatment (control and probiotic diet), the relationship between RGR
and ration size was analysed with a non-linear regression and GR curves were plotted.
The regressions were calculated to ﬁt the data within the range of the ration sizes (1% to
6%). The model describing the response was: (1) GR: y = y
), where y is the tank
average RGR, x is the ration ingested, and a, b are constants determined by the regression.
Furthermore, values of K1 in relation to ration size were plotted (KR curve) by using the
predicted values from the growth–ration model: (2) KR: y/x = (y0+ a(1 −bx))/x.
Animals 2021,11, 3451 7 of 21
2.5.3. Determination of Maintenance (Rm) and Optimal (Ropt) Rations
From the GR and KR curves, the maintenance (Rm) and optimal (Ropt) rations were
determined according to [
]. Rm is the feed intake that maintains the animal without any
change in its BW, and Ropt represents the feed intake that produces the greatest increase
in BW for the least feed intake, in other words, it determines optimal growth. Daily Rm
calculated from equation (1), when y is null, corresponds to the ration for which the RGR is
null. Ropt is identiﬁable on the GR curve as the ration for which the tangent crosses the
origin; Ropt was determined as the ration for which the KR curve reaches its maximum,
and it is equal to the value of x for which the ﬁrst order derivative of equation (2) is null
(i.e., when dK/dR = 0).
2.5.4. Scope for Growth (SFG)
The scope for growth (SFG) was deﬁned by [
] as “the difference between the energy
of the food an animal consumes and all other energy utilisations and losses”. Ref. [
able to demonstrate that the difference between any rations ingested, allowing growth and
the maintenance ration (Rm), gave a simple measurement of SFG. Indeed, only the part of
the feed allocation that is in excess of the Rm will be available for use in growth. In this
study we calculated the SFG as the difference between the digestible energy (DE) fraction
of the optimal ration (Ropt
DE) and the digestible energy fraction of the maintenance
ration (Rm ×DE) according to the following equation: SFG = DE ×(Ropt −Rm).
Statistical analyses were conducted using R software [
]. Prior to analysis, all data
were systematically checked for normal distribution and variance homogeneity. The per-
centage survival rates were normalized using an arcsine transformation before analysis.
Data on survival rates were tested using a one-way analysis of variance followed by a
Student’s multiple comparison t-test to determine differences among ration sizes and treat-
ments. The effects of treatments and ration size on the physiological parameters studied
were assessed by a two-way analysis of variances followed by a pairwise comparisons
using Fisher’s Protected Least Signiﬁcant Difference (PLSD). For each parameter, four
samples per tank were assayed. The effect of dietary treatment, ration size and their
interaction on BWf and RGR were tested using individual shrimp data by a two-way
nested analysis of variance in order to take into account a possible signiﬁcant random
tank effect. Indeed, variations between tanks can represent a random nuisance factor that
can lead to invalid conclusions if simple ANOVAs are used [
]. With the analysis of
variance model being mixed, the ration size and dietary treatment effects were tested from
a test of hypothesis using the random effect (tank effect) as error term, and individual
BWi was also used as a covariate in the model. The effect of dietary treatment, ration size
and their interaction on K1 and K2 were tested by a two-way analysis of variance with a
post hoc Student-Newman-Keuls test, whereby signiﬁcant differences occurred. Due to
limitations inherent to ANOVAs, the ration-size effect was further assessed within each
separate diet by non-parametric Kruskall–Wallis and a Mann–Whitney test was used to
determine differences between diet at each ration size. The GR- and KR-curves models
were determined and plotted for both experimental using Sigmaplot
software (SPSS Inc.).
Statistically signiﬁcant differences between experimental groups were reported at p< 0.05,
if not otherwise stated. Data are given as a tank means
standard deviation of triplicate
tanks (n = 3).
3.1. Survival, Growth and Nutritional Parameters
During the experiment, the shrimp consistently consumed all the feed provided
whatever the ration size, such that the amount of feed distributed was equal to the amount
of feed ingested. Zootechnical results at the end of the trial are shown in Table 3. The mean
ﬁnal survival rate was 81.6
15.0% without a diet or ration-size effect on this parameter
Animals 2021,11, 3451 8 of 21
even at a low feeding rate. The mean BWi was 10.93
1.78 g with no signiﬁcant difference
between dietary groups (control or probiotic diet). However, signiﬁcant differences due
to the random allocation of shrimps to ration size and treatment groups were detected
). Accordingly, BWi was used as a covariate to statistically assess the diet and ration-
size effect on BWf. RGR was used for a posteriori analysis to compare growth according to
treatment and ration size. No random tank effect was detected when individual RGR data
were used with a mixed ANOVA.
Survival, body-size and growth of L. stylirostris per diet group at each ration size. Mean
SD. For each parameter,
different letters within the same raw indicate signiﬁcant differences between diets within each ration size (Mann–Whitney
test, p< 0.05). Ration-size effects assessed by Kruskal–Wallis test.
Daily Ration Size
Survival (%) BWi (g) BWf (g) RGR (% BWi.day−1)
Control Probiotic Control Probiotic Control Probiotic Control Probiotic
183 ±17 a83 ±17 a11.55 ±0.10 a11.02 ±0.31 b11.25 ±0.07 11.06 ±0.40 −0.08 ±0.03 a0.03 ±0.03 b
275 ±12 a78 ±10 a10.70 ±1.42 a11.68 ±0.44 a11.64 ±1.54 13.47 ±0.22 0.33 ±0.02 a0.47 ±0.06 b
378 ±25 a83 ±17 a11.88 ±0.59 a10.70 ±0.31 b13.70 ±0.60 12.15 ±0.50 0.40 ±0.05 a0.56 ±0.02 b
489 ±19 a83 ±29 a11.15 ±0.41 a10.31 ±0.69 a12.03 ±1.55 11.81 ±0.67 0.45 ±0.08 a0.61 ±0.07 b
683 ±0a78 ±10 a10.84 ±1.19 a9.62 ±0.43 a12.07 ±1.01 11.06 ±0.98 0.45 ±0.04 a0.59 ±0.09 a
Ration-size effect n.s. n.s. n.s. ** - - *** ***
BMi, initial tank biomass; BWi, initial body-weight; BWf, final body-weight; RGR, relative growth-rate. n.s., non-significant; ** p< 0.01; *** p< 0.001.
Ration size had a signiﬁcant effect on RGR (Tables 3and 4; Figure 1a). A posteriori tests
indicated a signiﬁcant increase in RGR between 1, 2 and 3% rations for probiotic treatment
and between 1, 2 and 4% rations for control (Figure 1a). No statistical difference was found
between RGRs at 3%, 4% and 6% BM. The probiotic treatment resulted in a signiﬁcantly
higher RGR compared to the control at 1, 2, 3% (p< 0.01) and 4% (p< 0.05) but not at
6% ration size. Furthermore, a probiotic diet effect on BWf was detected when a mixed
ANOVA was applied (Table 4) along with a signiﬁcant random tank effect which was not
detected when a classic ANOVA was applied (Table 3).
Statistical signiﬁcance (p-values) of diet, ration size and their interaction, and of initial
body-weight and tank effects for each growth parameter determined based on type III sum of squares
from factorial ANOVA. When nested ANOVA were applied, ration size and treatment effects were
systematically tested from a test of hypothesis using the tank random effect as the error term. (n.s.,
not signiﬁcant; * p< 0.05; ** p< 0.01; *** p< 0.001).
Indices Diet Ration Size BWi Diet ×Ration Size TankEffect
BWf (1) (g) *** *** *** n.s. **
RGR (1) (% BWi.day−1)*** *** n.s. n.s. n.s.
K1 (2) *** *** n.a. * n.a.
K2 (2) ** *** n.a. n.s. n.a.
BWf, ﬁnal body-weight; BWi, initial body-weight; RGR, relative growth-rate; K1, gross feed conversion efﬁciency;
K2, net feed conversion efﬁciency.
Nested two-way analysis of variance with initial body-weight used as
covariate; (2) Two-way analysis of variance; n.a. not applicable.
Animals 2021,11, 3451 9 of 21
). Growth-ration (GR) curves determined per diet group from tank mean data and
feed conversion-efﬁciency ratio (KR) curves determined per diet group from respective GR
curves over the 27-day trial duration. Optimum ration size for growth (Ropt; broken lines) were
calculated from the ﬁrst order derivative of the KR equation (i.e., when (dRGR/x)/dT = 0). Data
shown as mean
SD, n = 3 with 4 shrimps/tank assessed. Different letters indicate signiﬁcant
differences between ration sizes within diets (Student-Newman-Keuls test; p< 0.05).
For both diets, the growth ration curves were found to ﬁt non-linear regressions which
plateaued at a ration size over 3%. The equations of the curves obtained for each treatment
are indicated in Figure 1a, and adjusted R-squares were over 99% for both regressions.
From these equations, Rm and Ropt were obtained by calculation. Rm and Ropt were both
lower, by 16.8% and 11.3%, respectively, in the probiotic compared to control group. As the
probiotic induced a parallel drop in the maintenance ration (Rm) and in the optimal ration
(Ropt), the scope for growth (SFG), which is the difference between the digestible energy of
the two rations, is similar for the control and probiotic group (Table 5).
Animals 2021,11, 3451 10 of 21
Gross (K1) and net (K2) feed conversion efﬁciency by L. stylirostris per test diet at the ration sizes tested. Mean
SD, n = 3. For each parameter, different letters within the same raw indicate signiﬁcant differences between diets
(Mann–Whitney test, p< 0.05). The ration size effects were assessed by a Kruskal–Wallis test (n.s., non-signiﬁcant; * p< 0.05;
** p< 0.01).
Daily Ration Size
K1 (%) K2 (%)
Control Probiotic Control Probiotic
1−7.87 ±2.61 a3.33 ±3.14 b- 83.33 ±45.3
2 16.88 ±0.75 a23.33 ±3.47 b36.30 ±1.88 a49.74 ±6.68 b
3 13.08 ±1.61 a18.53 ±0.19 b20.87 ±2.57 a27.25 ±0.27 b
4 11.25 ±2.85 a15.09 ±1.80 a15.62 ±3.96 a19.86 ±2.37 a
6 7.54 ±0.45 a9.73 ±2.01 a9.27 ±0.55 a11.59 ±02.40 a
Ration-size effect * ** * *
BMi, initial tank biomass.
Diet and ration size had a signiﬁcant effect on K1 (Table 4). The KR-curves (Figure 1b),
as derived from the GR-curve models, showed that, for both diets, K1 increased signiﬁcantly
from 1 to 2% ration size and progressively decreased thereafter with increasing ration size.
The probiotic compared to the control group had a signiﬁcantly higher K1 at 1, 2 and
3% but not at larger ration sizes (Table 6) and differences in K1 between diets decreased
with increasing ration sizes above Ropt (Figure 1b). K1 reached maximum values of 16.2%
and 23.2% at Ropt of 2.08% BM.day
and 1.88% BM.day
for the control and probiotic
treatment, respectively (Figure 1b).
Feed, gross energy and digestible energy intake per diet group at the calculated maintenance and optimal feed
ration size. Estimate of the “scope for growth” and predicted growth-rate of shrimps at optimal ration size.
Feed and Growth
Maintenance Ration (Rm) Optimal Ration (Ropt)
Control Probiotic Control Probiotic
Feed intake (g.kg−1.day−1)11.3 9.4 21.3 18.9
GE intake (a) (kcal.kg−1.day−1)50.9 42.3 95.9 85.1
DE intake (b) (kcal.kg−1.day−1)38.1 31.7 71.9 63.8
SFG (kcal) 33.8 32.1
GR (c) (g.kg−1.day−1)3.3 4.5
GE, gross energy; DE, digestible energy; SFG, scope for growth; GR, growth rate.
based on basal diet GE = 4502 kcal.kg
based on basal diet DE = 3376 kcal.kg−1(Table 1); (c) predicted optimal growth rate at Ropt.
The K2 values were not calculated for the control group fed at the ration size of 1%, as
the mean relative growth rate was negative. Additionally, K2 was found to signiﬁcantly
decrease with increasing ration sizes (Tables 4and 6) and was signiﬁcantly higher at the
2% compared to a 3% ration size in both diets. The probiotic group exhibited signiﬁcantly
higher K2 values than the control at feeding rates of 2% and 3% BM.day
. Finally, aside
for K1, no signiﬁcant interaction between diet and ration size were detected for any of the
variables tested (Table 4).
3.2. Biochemical Parameters
3.2.1. Digestive Enzymes
There were signiﬁcant diet and ration-size effects without interactions on
speciﬁc activity in the digestive gland (Table 7). The speciﬁc activity of
overall with increasing ration size in both diet groups; and signiﬁcantly higher activities
were measured in the probiotic compared to the control groups at ration sizes of 1%, 2%
and 3% (Table 7; Figure 2a). There was a signiﬁcant diet effect and diet
interaction on the speciﬁc activity of trypsin in the digestive gland (Table 7). In the control
group, trypsin activity decreased with increasing ration size, reaching levels twice lower
when fed at 6% compared 1% BM.day
(Table 8). In comparison, trypsin activity was not
Animals 2021,11, 3451 11 of 21
affected by ration size (p> 0.05), instead remaining consistently high in the probiotic group.
Variations in the amylase/trypsin ratio were not explained by any of the parameters tested
(Table 7). Only one signiﬁcant difference between treatments was found at 1% ration size,
where probiotic fed shrimps showed higher values for this ratio (Table 8).
-amylase activity, (
) glycogen level and (
) glucose level in digestive gland;
level in haemolymph of L. stylirostris according to ration size and dietary treatment at the trial’s end-
point. Data shown as mean ±SD, n = 3 with 4 shrimps/tank assessed. Asterisk indicate signiﬁcant
differences between diets at each ration size (2-way ANOVA, PLSD; * p< 0.05, ** p< 0.01).
Statistical signiﬁcance of diet, ration size and their interaction for each biochemical parameter
measured in the (
) digestive gland and (
) haemolymph (2-way ANOVA; n.s., non-signiﬁcant; * p<
0.05, ** p< 0.01, *** p< 0.001).
Parameter Diet Ration Size Diet ×Ration Size
(a) Digestive Gland
α-amylase activity (U.mgprot−1)*** *** n.s.
Trypsine activity (U.mgprot −1)** n.s. *
α-amylase/trypsine n.s. n.s. n.s.
Glucose (mg.gorgan−1)n.s. ** n.s.
Glycogene (mg.gorgan−1)** * n.s.
TAS (µmol.gorgan−1)*** *** n.s.
Glucose (mg.mL−1)* * n.s.
TAS (µmol.mL−1)n.s. * n.s.
TAS, total antioxidant status.
Animals 2021,11, 3451 12 of 21
Speciﬁc activities of digestive enzymes in the digestive gland of L. stylirostris per test diets at each ration size
tested. Values given as mean
SD with n = 3. For each parameter, different letters within the same raw indicate signiﬁcant
differences between diets by pairwise comparisons using Fisher’s Protected Least Signiﬁcant Difference (PLSD). The
ration-size effects were assessed by a Kruskal–Wallis test (n.s., non-signiﬁcant; ** p< 0.01; *** p< 0.001).
Control Probiotic Control Probiotic Control Probiotic
1 4.03 ±0.12 a5.77 ±0.29 b0.38 ±0.04 a0.33 ±0.04 a11.96 ±1.16 a18.50 ±2.26 b
2 4.56 ±0.27 a5.52 ±0.11 b0.30 ±0.03 a0.38 ±0.05 a16.00 ±1.80 a16.29 ±0.73 a
3 3.62 ±0.26 a4.43 ±0.20 b0.31 ±0.03 a0.27 ±0.02 a11.99 ±2.47 a16.91 ±1.45 a
4 2.63 ±0.54 a3.05 ±0.12 a0.22 ±0.04 a0.23 ±0.01 a12.69 ±1.36 a13.40 ±0.66 a
6 3.19 ±0.31 a3.90 ±0.31 a0.18 ±0.02 a0.32 ±0.06 b17.80 ±2.61 a15.60 ±4.63 a
effect *** ** ** n.s. n.s. n.s.
BMi, initial tank biomass.
3.2.2. Glucose and Glycogen Content
There were signiﬁcant diet and ration-size effects on glycogen content in the digestive
gland (Table 7). The probiotic group exhibited signiﬁcantly higher levels compared to
the control at ration sizes of 1%, 2% and 3% while no difference was detected at higher
feeding rates (Figure 2b). Within diets, glycogen content statistically increased by up to
4% and up to 2% in the control and probiotic group, respectively. Free glucose in the
digestive gland was also signiﬁcantly higher in the probiotic compared to the control group
at ration sizes of 1, 2 and 3% (Figure 2c) with levels remaining unchanged across ration
sizes. In comparison, in the control group, free glucose in the digestive gland increased
between ration sizes of 1% and 3%, 4%, and 6%; thereby reaching the consistently high
levels measured across the probiotic group at a 4% ration size and above.
In the haemolymph, free glucose slightly, but not signiﬁcantly, increased up to the ra-
tion sizes of 3% and 2% in the control and probiotic groups, respectively (
and reached an apparent saturation level close to 1mg.mL
in both diet groups. Com-
pared to the control, the probiotic group had a signiﬁcantly higher haemolymph glucose
concentration at a ration size of 1% and 2%.
3.2.3. Total Antioxidant Status
There were signiﬁcant diet and ration-size effects on TAS of the digestive gland; and
a signiﬁcant ration-size effect on TAS of the haemolymph (Table 7) without interactions.
In the digestive gland, TAS decreased overall with an increased ration size (Figure 3a)
reaching signiﬁcantly lower values at 3%, 4% and 6% compared to 1% and 2% in both diets.
On the other-hand, haemolymph TAS remained consistent across ration sizes (Figure 3b).
With regard to the diet effect, TAS levels in both the digestive gland and haemolymph were
signiﬁcantly higher in the probiotic compared to the control group across all ration sizes
tested, except at 6%.
Animals 2021,11, 3451 13 of 21
Total Antioxidant Status (TAS) in (
) the digestive gland and (
) haemolymph of shrimps
from each treatment according to each ration size at the trial’s end-point. Mean
SD, n = 3 with 4
shrimps/tank assessed. Asterisk indicate signiﬁcant differences between diets at each ration size
(2-way ANOVA, PLSD; * p< 0.05).
4.1. GR-Curves and Nutritional Parameters in the Control Diet
The two growth-ration (GR) models established in this study for sub-adults L. stylirostris
fed a control or probiotic diet were each based on ﬁve ﬁxed ration sizes that were restricted
and systematically fully ingested. This is of particular importance as a precise measure of
feed intake is essential to the accuracy of this type of model and is particularly difﬁcult to
measure in shrimp, owing to their slow-feeding behaviour.
The GR-curves obtained from the mean RGR values per tank (Figure 1a) were used
along with the KR-curves, expressing the relationship between ration size and gross feed
conversion efﬁciency, K1 (Figure 1b), to determine the daily nutritional requirements of
the shrimp for maintenance (Rm) and optimal growth (Ropt; [
]). The fundamental GR-
and KR-relationships are overall well documented in ﬁsh [
] but have only rarely
been investigated in shrimp [
]. Ref. [
] used various daily ration sizes to estimate
the daily protein requirements of juvenile L. vannamei. The GR-relation in L. stylirostris
is similar in appearance to those described for some ﬁshes [
] and can be ﬁtted to
a second-order polynomial regression. However, the literature is equivocal with respect
to the shape of the feeding relationship of growth in marine ﬁsh. Both linear [
non-linear (asymptotic; [
]) relationships are described, and this may also pertain to the
range of ration sizes tested. In this study, digestible Rm of the control diet was equal to 9 g
shrimp (Table 5) which corresponds to a digestible energy (DE) of 38.14
shrimp (159 kJ.day
shrimp). This value, which is an estimate of
the maintenance energy requirement, is 30% greater than that of the fasting heat production
or standard metabolism determined by measuring oxygen consumption of fasting shrimp
L. stylirostris (116
7.7 kJ. day
shrimp; BW = 10.6
0.4 g; n = 9, 28
result is consistent because it is well accepted that the maintenance energy requirements
are between 30 and 60% greater than for basal metabolism [
] and conﬁrms the accuracy
of the measurement of Rm from the GR- and KR-models for shrimp. Standard metabolism
requirements are typically estimated directly, by measuring oxygen consumption rates and
ammonia excretion. This approach is analytically precise but requires the conﬁnement of
the shrimp in special apparatus which may affect the animal’s response and the results are
usually recorded over short time-periods (24 h to 48 h). Under such conditions, metabolic
requirements may be over- or underestimated if handling stress [
] and moulting
] are not considered. In the present study, the maintenance energy requirement
and Rm were estimated from an experimental period covering several weeks, hence, fully
integrating various events associated with the shrimp’s biological rhythms and cycle
Animals 2021,11, 3451 14 of 21
(moulting cycle, feeding, growth, activity and rest) as well as “real-life” ﬂuctuations in
environmental, social and zootechnical parameters.
The maximum gross conversion efﬁciency (K1) of sub-adult L. stylirostris fed the con-
trol diet was 16.88
0.75% under our experimental conditions. This value is approximately
half of that measured value in the same experimental conditions with younger L. stylirostris
0.4 g, n = 150; K1 = 40%; [
]). The lower K1 in this study may reﬂect the weaker
capacity of larger L. stylirostris specimens to transform feed into body tissues compared to
smaller ones [
]. We also observed a gradual decline in K1 at ration sizes greater than Ropt,
which may be the result of a quicker digestif transit when feed intake increases. Similarly,
the net conversion efﬁciency (K2) of the control diet was higher at a ratio of 2% BWi.day
and then decreased at a ratio of 3% BWi.day
and more. Therefore, shrimp seem to require
less feed per unit of weight-gain when the ration size is restricted as previously observed
in P. monodon [
]. The maximum K2 obtained in our study (36.3
1.9%) was within the
range of values obtained for the larval stage and juveniles of other crustaceans [69–71].
4.2. Effects of the Probiotic on Growth and Nutritional Parameters
Probiotic studies in shrimps more often focus on health, increased disease resistance
and related modes of action while their effect on growth remains overall less addressed.
Amongst others, Refs. [
] investigated the effect of Lactobacillus sp., Bacillus sp.
photosynthetic bacteria (Rhodobacter sphaeroides) and Gram-negative bacteria (Enterobacter
hormaechei) on the growth of M. rosenbergii,Fenneropenaeus indicus and L. vannamei. In
their experiments, shrimps were fed ad libitum and the effect of the probiotic were deter-
mined based on growth rate, feed conversion ratio and digestive enzyme activities such
as protease, lipase and amylase. The authors usually linked the observed beneﬁt of the
probiotic on growth to enhanced digestion and nutrients absorption and to higher activities
of digestive enzymes. In the present study, (Figure 1), we examined the effect of a constant
daily intake of P. acidilactici on the GR and KR-relationships initially proposed by [
GR-curves obtained for each dietary treatment were parallel with the probiotic curve above.
Dietary probiotic intake at a dose of 4
shrimp therefore appeared
to promote growth compared to the same non-supplemented diet for a given ration size.
Accordingly, based on statistical analysis, RGR was found to plateau at a lower ration size
for the probiotic (3% BM.day
) compared to the control (4% BM.day
), suggesting that
maximum growth is reached at a smaller ration size when the diet is supplemented with
P. acidilactici. Moreover, and interestingly, shrimps fed with the probiotic diets showed
reduced Rm and Ropt and increased K1 and K2. At Ropt and despite a smaller Ropt,
the probiotic diet resulted in a RGR increase of over 36% compared to the control (4.5
and 3.3 g.kg
, respectively). Accordingly, K1 max was improved by
38% and K2 to Ropt by 37% in the probiotic compared to the control diet, which taken
together indicate a better transformation of the feed into shrimp growth. Finally, the present
data show that at a 1% ration size, shrimps fed the probiotic diet did not lose weight while
control shrimps did (RGR = 0.3 g.kg
and -0.08 g.kg
for the probiotic and
control groups, respectively).
Taken together, these results indicate that shrimps supplemented with probiotic
require less feed to reach maintenance and optimal growth and that growth at Ropt is also
superior, suggesting enhanced feed utilisation. Two other hypotheses may also explain
these results. Firstly, the probiotic could provide some growth factors or essential nutrients
favouring growth, as demonstrated with other probiotic strains [
]. Second, the probiotic
could result in a decreased metabolic demand as suggested in this study by the lower
feed requirement for maintenance and by the fact that probiotic-fed shrimps did not lose
body-weight at 1% BM.day
, unlike in the control. This could be evaluated by comparing
oxygen consumption rate of shrimps under both dietary regimes. It must be noted that
the enhanced growth and nutritional performance as a result of the probiotic intake were
documented under ﬁxed, restricted rations (below satiation), and over a short grow-out
period under laboratory conditions. Further trials must therefore be performed under
Animals 2021,11, 3451 15 of 21
commercial conditions and through feed management over a longer timeframe to assess
the potential contribution of the probiotic at farm-level.
4.3. Dietary Carbohydrates (CBH) Utilization and the Effect of Probiotic
Carbohydrates are an important energy source in shrimp diets favouring growth and
fat deposition [
]. However, CBH are not efﬁciently utilized by shrimp which possess
low carbohydrate digestion capacity and a low plasma glucose regulatory ability [
applying a range of ﬁxed and restricted rations, the study assessed the effect of a graded
level of CBH intake on CBH utilization with or without probiotic supplementation.
Glycogen and free glucose values measured in this study were within the range
previously reported for L. vannamei [
]. In the control group, the glycogen content of the
digestive gland tended to increase with increasing ration size until apparent saturation
above 3% BM.day
with a similar trend for free glucose level. In comparison, probiotic fed
shrimps, overall, showed higher and more consistent glucose and glycogen levels. Ref. [
reported the saturation of the glycogen level in the digestive gland for L. stylirostris fed
over 21% dietary carbohydrates (CBH); which they suggested as the maximum dietary
CBH level and apparent maximum capacity for dietary CBH utilization. However, in
their studies, feed intake was not measured, making quantitative requirements for CBH
impossible to determine. In an L. vannamei study, the same authors [
] reported the
saturation curve of glycogen level in the digestive gland observed for diets with dietary
CBH levels higher than 33%. In the present study, and based on glycogen levels in the
digestive gland, it can be estimated that the control shrimps reached a maximum capacity
to use CBH at a feed intake close to 4% BM.day
, compared to a lower ration size of 2% to
when supplemented with the probiotic. It can therefore be hypothesized that
probiotic supplementation resulted in a more efﬁcient use of available dietary CBH until
saturation was reached. Once saturation is reached at ration sizes above 3% BM.day
probiotic was fond to have no further discernible effects on CBH. This is supported by the
absence of a signiﬁcant probiotic effect on the glycogen and glucose levels of the digestive
gland at these higher ration sizes, where a reduced probiotic effect on growth and gross
feed efﬁciency was also observed.
In shrimps, once feed is ingested, starch is ﬁrst processed by
-amylase to produce
oligosaccharides and then glucose through
-glucosidase. This system gives rise to the slow
liberation of glucose into the blood and explains why starch is viewed as an efﬁcient CBH
source in shrimp feeds [
]. Refs. [
] showed that in L. stylirostris and in L. vannamei,
the hydrolysis of dietary starch by
-amylase could be limited by dietary CBH, and that
-glucosidase was directly related to but not limited by dietary CBH level. These authors
also reported a saturation of glycogen and
-amylase speciﬁc activity above the same level
of dietary CBH [
]. In this study, the higher glycogen and glucose contents of the digestive
gland in probiotic-fed shrimps fed with a lower ration size (1, 2 and 3% BM.day
associated with a higher
-amylase speciﬁc activity and haemolymph glycemia (1% and
). This strongly supports the hypothesis of a better utilisation of dietary CBH
through P. acidilactici supplementation via the stimulation of
-amylase speciﬁc activity.
Moreover, as for amylase, trypsin activity decreased with an increasing feeding rate which
may, together, partly explain the lower feed efﬁciency recorded at larger ration sizes. Finally,
the stable amylase/trypsin ratio measured across ration sizes in this study was consistent
with values previously reported in L. vannamei [
] and with these authors’ suggestion
that its alteration would mainly be linked to changes in dietary composition, as previously
Several authors [
] have assumed that probiotics may stimulate the production
of endogenous enzymes and it is possible that the probiotic produces substances, such as
vitamins, which will speciﬁcally inﬂuence some digestive enzyme activities. For instance,
vitamin C has been found to increase the activity of amylase [
] in shrimps, and growth
factors presented in some feed ingredients have increased speciﬁc activities of amylase,
trypsin and total proteases in Marsupenaeus japonicus [
]. Moreover, in an unpublished
Animals 2021,11, 3451 16 of 21
study, Ref. [
] was able to show that some extracellular protein products secreted by
Lactobacillus farciminis MA27/6R and Lactobacillus rhamnosus MA27/6B were able to speciﬁ-
cally enhance trypsin and
-amylase activity in Artemia. Besides, dietary probiotics may
also exert their inﬂuence on digestive functions indirectly by modulating the composition
of the endogenous intestinal microbiota [
], which has not been addressed here. This
hypothesis is further supported by recent investigations regarding the functional role of the
intestinal microbiota of shrimp and the link with the nutritional and phycological status of
the animal [88,89].
4.4. A Link between Carbohydrate Metabolism and the Antioxidant Status
] previously documented that an increased concentration of liver-glycogen
content, from increased dietary CBH intake, resulted in a decreased activity of the main
antioxidant enzymes superoxide dismutase and catalase. This apparent link between
CBH metabolism and the activity of antioxidant enzymes was tentatively explained by
free glucose and simple sugars as a direct scavenger of OH-radicals [
] as well as a
potent stimulant of the pentose phosphate shunt that regenerates NAD
to NADPH hence,
affecting the cellular redox status .
In a prior study, we reported decreases in superoxide dismutase and catalase activity
in L. stylirostris fed P. acidilactici compared to non-supplemented shrimps [
]. In the
present and subsequent study, Total Antioxidant Status (TAS) was measured to determine
whether a potential probiotic effect on dietary CBH utilization would be concomitant with
an altered antioxidant status. In the digestive gland, TAS decreased for greater ration sizes
(3, 4 and 6% BM.day
) with mean values similar to those previously reported (12.88
) in L. stylirostris fed ad libitum [
]. It has been demonstrated that feed intake
and digestion increase the aerobic metabolism of L. stylirostris [
], while postprandial
metabolism is known to rise with increased feed ingestion in isopod
the postprandial metabolism increases with the amount of feed consumed, the production
of ROS is also expected to increase and, therefore, the TAS level to decrease as has been
observed here. In crustaceans, the mobilization of antioxidant defenses may be particu-
larly evident and critical in the lipid-storing digestive gland [
] against the risk of lipid
peroxidation at greater ration sizes.
Finally, the dietary probiotic was found to signiﬁcantly increase TAS level in both
the haemolymph and digestive gland (except for at the highest ration size of 6%), in
line with previous reports [
]. The positive effect of P. acidilactici on the antioxidant
status of the shrimp could be the result of the better use of dietary CBH in probiotic-fed
shrimp, as suggested by the work of [
] and as is overall supported by the data presented
in this study. However, the mode of actions of probiotics are overall very diverse and
a combination of several action mechanisms is expected to be involved for any given
(generalist) probiotic [
]. For instance, the probiotic effect on antioxidant status might
also be based on the antioxidant properties of the strain, as observed for other lactic acid
bacteria used as probiotic .
In conclusion, the growth-ration method applied in this study was effective at doc-
umenting a clear contribution of a dietary probiotic on the growth and feed efﬁciency of
sub-adults L. stylirostris fed a ﬁxed ration under controlled conditions. The optimal ration
(Ropt) was 1.88% BM.day
in the probiotic group compared to 2.08% BM.day
control, while RGR and Ropt were also improved in the probiotic group. The experimental
approach combined individual tagging to statistically assess the effect of ration size on
growth, followed by establishing the GR-relation that links ration size to the mean relative
growth-rate per tank. This combined approach is proposed as a useful way of understand-
ing how a feed additive can inﬂuence shrimp growth and feed efﬁciency. The effect of
dietary P. acidilactici MA18/5M on
-amylase activity and CBH utilization and metabolism,
and its link to the shrimp antioxidant status should be further studied. This study warrants
Animals 2021,11, 3451 17 of 21
further research on the contribution of this probiotic under commercial (feed) management
over the grow-out cycle, as it presents the prospect of optimizing dietary formulation, as
well as the biological and economic efﬁciency of the shrimp-farming industry.
Conceptualization, M.C. and L.C.; methodology, M.C. and L.C.; software,
M.C.; validation, L.C.; formal analysis, M.C.; investigation, M.C., P.L. and L.C.; resources, L.C.;
data curation, M.C.; writing—original draft preparation, M.C.; writing—review and editing, E.L.;
visualization, E.L.; supervision, L.C.; project administration, L.C.; funding acquisition, M.C. and L.C.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement:
Ethical review and approval were waived for this study
performed on a marine invertebrate other than cephalopods (shrimp) which do not require Ethical
review and approval according to EU directive 2010/63/EU (22 September 2010).
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
The research was part of M.C. PhD project which was ﬁnancially supported by
Lallemand SAS with in-kind contribution from IFREMER. The authors thank the IFREMER technical
staff of the experimental facilities at St Vincent, New Caledonia for their help.
Conﬂicts of Interest:
The experiment was in part supported by Lallemand SAS with the aim of
investigating the effect of a commercial probiotic product and where M.C. and E.L. are employed.
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