General and Comparative Endocrinology 188 (2013) 297–302

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General and Comparative Endocrinology

journal homepage: www.elsevier.com/locate/ygcen

Communication in Genomics and Proteomics

Effects of probiotic administration on zebrafish development and reproduction

O. Carnevali a,b,⇑, M.A. Avella a,1, G. Gioacchini a
a
Department of Life Sciences, Università Politecnica delle Marche, Ancona, Italy
b
Istituto Nazionale Biostrutture e Biosistemi, Roma, Italy

a r t i c l e i n f o a b s t r a c t

Article history: As the consumption of probiotics increases worldwide, scientists focus on identifying bacterial strains
Available online 13 March 2013 able to improve human life quality and evidence the biological pathways affected by probiotic treatment.
In this review, some recent observations on the effects of changes of microbiota on zebrafish metabolism
Keywords: were discussed.
Danio rerio In addition, the effects of Lactobacillus rhamnosus – a component of the human gut microflora – as a diet
Lactobacillus rhamnosus supplement on Danio rerio were presented. When administered chronically, L. rhamnosus may affect lar-
Bone development
val development and the physiology of reproductive system in the zebrafish model. It was hypothesized
Gonadal differentiation
Gonadal function
exogenous L. rhamnosus accelerates larval growth and backbone development by acting on insulin-like
growth factors-I (igfI) and -II (igfII), peroxisome proliferator activated receptors-a and -b, (ppara,b) vita-
min D receptor-a (vdra) and retinoic acid receptor-c (rarc). Gonadal differentiation was anticipated at
6 weeks together with a higher expression of gnrh3 at the larval stage when L. rhamnosus was adminis-
tered throughout development. Moreover, brood stock alimented with a L. rhamnosus-supplemented diet
showed better reproductive performances as per follicles development, ovulated oocytes quantification
and embryos quality. A plausible involvement of factors such as leptin, and kiss1 and 2 in the improve-
ments was concluded. The observations made on the physiology of female reproduction were correlated
with the gene expression of a gigantic number of factors as the aromatase cytochrome p 19 (cyp19a), the
vitellogenin (vtg) and the a isoform of the E2 receptor (era), luteinizing hormone receptor (lhr), 20-b
hydroxysteroid dehydrogenase (20b-hsd), membrane progesterone receptors a and b, cyclin B, acti-
vinbA1, smad2, transforming growth factor b1 (tgfb1), growth differentiation factor9 (gdf9) and bone
morphogenetic protein15 (bmp15.) A model in which the exogenous L. rhamnosus in the digestive tract
of zebrafish from the first days of life through sexual maturation positively influences the physiological
performances of zebrafish was evidenced and a number of pathways that might be influenced by the
presence of this human probiotic strain were proposed.
Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction development. In addition, zebrafish and human genomes have

Over the last past 20 years, the zebrafish (Danio rerio) has at-
tracted considerable attention as a pre-eminent vertebrate model
for studying genetics and development (Grunwald and Eisen,
2002), and more recently, for understanding human diseases and
for the screening of therapeutic drugs (Keller and Murtha, 2004;
Mathias et al., 2012; Penberthy et al., 2002; Sumanas and Lin,
2004). The characteristics that have contributed to its popularity
as a model of human disease and development are: ease to produce
and maintain in laboratory conditions, small size, rapid develop-
ment and generation time, optical transparency during early
⇑ Corresponding author. Address: Department of Life and Environmental Sci-
ences, Università Politecnica delle Marche, via Brecce bianche, 60131 Ancona, Italy.
E-mail address: o.carnevali@univpm.it (O. Carnevali).
1
Current address: National Institute of Diabetes and Digestive and Kidney
Diseases, NIH, Bethesda, USA.
been shown to share extensive conserved syntenic fragments and
many zebrafish genes and their human homolog display structural
and functional genetic similarities to humans (Barbazuk et al.,
2000) and finally, its genome sequence are further refined and
available on web (http://www.ncbi.nlm.nih.gov/genome/guide/
zebrafish/).
Given the importance of this experimental model, zebrafish was
used to study the effects of the Lactobacillus rhamnosus, a probiotic
registered for human consumption, on the intestinal microbiota,
gonadal development and reproduction of zebrafish.
The genus Lactobacillus produce and secretes numerous sub-
stances; these may be grouped in substances anchored to the lipid
membrane, to cell wall, cell membrane by N- or C-terminus,
released into the extracellular medium or be part of the cellular
membrane appendance (Lebeer et al., 2008). Transcriptomic and
proteomic studies evidenced a large number of neo secreted pro-
tein by Lactobacillus, and these effector molecules influence the

0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ygcen.2013.02.022
298 O. Carnevali et al. / General and Comparative Endocrinology 188 (2013) 297–302

host organism at different levels, including the inhibition of were observed in respect to microbial richness (P1=4 0.105), diver-
intestinal pathogens (Eijsink et al., 2002), mucosal integrity (Seth sity (P1=4 0.112) and evenness (P1=4 0.138).
and Yan, 2008; Yan et al., 2007), immune system modulation Bacterial populations on the gastrointestine affected individual
(Smits et al., 2005), metabolic interactions (Marco et al., 2006; phylotypes either by suppressing them or by stimulating the pres-
Semova et al., 2012), cell growth (Schlee et al., 2008) and reproduc- ence of certain phylotypes that were absent or no detectable in the
tion (Gioacchini et al., 2010a,b,c; Gioacchini et al., 2011; Gioacchini control. In zebrafish, the treatment with L. rhamnosus increased the
et al., 2012; Gioacchini et al., 2013; Giorgini et al., 2010). presence of Streptococcus thermophilus causing a clear shift in
microbial communities (Gioacchini et al., 2012).
The PCR-DGGE analysis in the ovaries showed the absence of L.
2. Zebrafish gut microbiota rhamnosus and any detectable bacterial rRNA in the gonad indicat-
ing that the probiotic does not affect directly the ovary but operate
The knowledge of how gut microbial communities gather and by systemic way (Gioacchini et al., 2012).
cooperate with vertebrate hosts is largely derived from a few lab-
oratory model species including zebrafish (Camp et al., 2009;
3. Effects of probiotic on growth and calcification
Cheesman and Guillemin, 2007). In fact, zebrafish has been exten-
sively used to study vertebrate development and physiology; only
Zebrafish raised with probiotic-supplemented diet showed fas-
recently it has been recognized as a model for studying host–mic-
ter development, with earlier onset of backbone calcification (Avel-
robiota interactions. At this purpose, lately a method for zebrafish
la et al., 2012).
gnotobiotic husbandry, to study host responses to the gut microbi-
Previous studies (Hooper and Gordon, 2001; Hooper et al.,
ota was optimized (Rawls et al., 2004).
2002) showed that beneficial microbes led an organism to more
The first insights into the association of gut microbiota from
efficiently use diet-derived energy sources, leading to growth
zebrafish reared in laboratory were obtained by sequencing li-
improvement (Dimitroglou et al., 2011). The administration of L.
braries of bacterial 16S rRNA genes (Bates et al., 2006; Brugman
rhamnosus to zebrafish larvae induced the presence of this bacte-
et al., 2009; Rawls et al., 2006). These authors evidenced that zeb-
rial in the intestine and a plausible increased of Lactobacillus’
rafish gut microbiota was rich in the bacterial phylum Proteobac-
metabolites in the intestine that, by a systemic diffusion, may exert
teria, with the phyla Firmicutes and Fusobacteria during both
its influence on the expression of specific genes (Avella et al.,
larval and adult stages. Recently, Roeselers et al. (2011) evidenced
2012).
that the gut microbiota of laboratory-reared zebrafish has similar
Rawls et al. (2004) evidenced the ability of the microbiota to
composition to that of zebrafish collected from their natural habi-
modulate the transcription of 212 genes, 8 of which were impli-
tat. The presence of bacterial members shared in caught and
cated in the promotion of nutrient metabolism. Avella et al.
domesticated zebrafish in different locations, suggested a selection
(2012) recently correlated the presence of L. rhamnosus in the zeb-
of taxa by intestinal habitat with a realization of a core gut micro-
rafish digestive tract with a modulation of the expression of several
biota (Roeselers et al., 2011). The genera present in the gastrointes-
genes involved in larval development. The growth-related factors,
tinal tract seem to be those which can survive and multiply in the
the insulin-like growth factors I and II (igfI and igfII) showed higher
intestinal tract (Ring and Strm, 1995).
gene expression in treated fish, while myostatin (mstn) levels were
The bacterial genera display day-to-day fluctuations; in fact, the
significantly reduced by probiotic administration. Changes of igfs
bacterial composition may vary with salinity, antibiotics and chro-
and mstn were correlated with higher zebrafish growth. The igfI
mic oxide (Ring and Strm, 1995). However, the overall diversity of
and II up-regulation (Avella et al., 2012) found in zebrafish admin-
bacterial communities may vary as a function of diet and environ-
istered with L. rhamnosus was found concomitant with an antici-
ment (Sullam et al., 2012; Wong and Rawls, 2012). Using gnotobi-
pated backbone development. Together with these changes, an
otic zebrafish, Semova et al. (2012) evidenced that bacterial
increase of vdra and rarc gene expression was observed. The li-
diversity and richness were markedly higher in the intestine of
gands of these two receptors (vitamin D and retinoic acid) are
fed fish in respect to starved ones, indicating that feeding results
key factors for morphogenesis and chondrogenesis process (Men-
in gut-specific enrichment of bacterial species in zebrafish. Semova
delsohn et al., 1994a,b,c). The vitamin D stimulates calcium
et al. (2012) also evidenced that microbiota composition might
absorption and retention (Saggese et al., 2002), while the igfs plays
influence energy balance of the host by the up-regulation of fatty
a role as main determinants of backbone calcification and bone
acid uptake and lipid droplets formation.
mass accretion, controlling muscle and skeletal cell growth and
Zhou et al. (2012) applied 10 Lactobacillus strains via the food to
division. A number of vitamins and fatty acids are naturally re-
zebrafish to test the adhesive capabilities on the intestine walls
leased by Lactobacillus spp., these products are able to bind differ-
using partial rpoB gene DGGE analysis, clone libraries and scanning
ent nuclear receptors including the vdr, rar (Chawla et al., 2001;
electron microscopy. Changes of Lactobacilli levels in the gastroin-
Narva et al., 2004; Teusink and Smid, 2006) and on ppara and b
testinal (GI) tract of probiotic-treated fish revealed that the adhe-
both involved in skeletal development (Burdick et al., 2006). The
sive capabilities of Lactobacilli varied greatly; the L. buchneri, L.
study on calcification evidenced a more extensive calcification of
plantarum, L. brevis and L. rhamnosus were identified as highly
centra in group treated with probiotics, indicating a faster back-
adhesive gut strains. Microbial changes in the GI were also ob-
bone calcification (Avella et al., 2012).
served when L. rhamnosus501TM was administered, the same spe-
cies was present in the intestine of all probiotic-treated group,
while it was absent in all replicates from the control group. The 4. Effects of L. rhamnous on gonadal differentiation and
PCR-DGGE fingerprints analysis revealed that the impact of the reproduction
administration of the probiotic had a clear and typical effect on
the microbial communities, causing a shift in microbial finger- Study performed in the zebrafish intestine evidenced that, in the
prints, and low similarity of approximately 50% between the developing intestine, the resident intestinal bacteria influence epi-
groups (Gioacchini et al., 2012). thelial cells by the enhancing b-catenin stability and promoting cell
Although the large dissimilarity between the groups, no signif- proliferation (van der Flier and Clevers, 2009). b-catenin is under the
icant differences were observed in terms of microbial community control of wnt signaling, its protein is a transcription factor that
and ecological parameters. However, trends toward differences once translocated into the nucleus interacts with co-activators, such
 O. Carnevali et al. / General and Comparative Endocrinology 188 (2013) 297–302
as the transcription factor tcf4, to turn on transcription of prolifera-
tive target genes, including c-myc and sox9 (Blache et al., 2004; van
de Wetering et al., 2002). Sox9 is an autosomic gene also involved in
chromosomal control of testis differentiation. A modulation of sox9
may be also hypothesized in the gonads where the administration of
probiotic induced an increase of testis differentiation (Avella et al.,
2012).
Zebrafish is a protogynous hermaphrodite species (Takahashi,
1977) that first develops ovary-like gonads. Bisexual differentia-
tion takes place when ovaries undergo differentiation, first turning
into an intermediate phase named altered ovary and finally into
testes (Maack and Segner, 2003). Avella et al. (2012) studied the ef-
fects of L. rhamnosus administered from the first feeding day
through the following 9 weeks, on gonadal differentiation and
sex ratio.
In the 3 weeks post fertilization (pf) untreated animals, the
majority of fish still showed undifferentiated gonad, only 20% of
specimens showed ovary-like gonads; the first testis appeared at
9 weeks pf. When L. rhamnosus was administered, at 3 weeks pf,
the treated population presented 10% of individuals with an undif-
ferentiated gonads, 20% with ovary-like gonads, 50% altered ova-
ries and strikingly, 20% already presented testis. At 6 weeks pf,
fish from control group showed 20% of undifferentiated gonad
and 80% of ovary-like gonads. In the group administered with pro-
biotic, 20% showed ovary-like gonads, 60% presented altered ova-
ries and 20% testis. Nine weeks pf, in the control group, 93%
specimens were females and only 7% males, while in the probiotic
group, 55% were females and 45% males, evidencing a clear effect
of L. rhamnosus also on sex ratio (Avella et al., 2012). However,
the sexual behavior in both groups appeared at 10 weeks pf, and
recently, the probiotic ability to affect reproductive physiology
was also studied (Gioacchini et al., 2010a,b,c; Gioacchini et al.,
2011; Gioacchini et al., 2012; Gioacchini et al., 2013; Giorgini
et al., 2010).
It is well known that nutrition affects all reproductive events,
the achievement of the size needed to initiate the reproductive
processes, normally referred to as puberty and gametogenesis,
are closely linked with the availability of energy in both males
and females. The association between nutrition and reproduction
is important to ensure that the reproductive event will be synchro-
nized with the nutrient availability to guarantee the survival of
new progeny (Scaramuzzi et al., 2006).
A number of studies are available on the effects of the probiotic,
L. rhamnosus, administered at 106 CFU g 1, on zebrafish reproduc-
tion (Gioacchini et al., 2010a,b,c; Gioacchini et al., 2011; Gioacchini
et al., 2012; Gioacchini et al., 2013; Giorgini et al., 2010). First of all
it was reported that after a 10 days-treatment, L. rhamnosus in-
duced a significant increase of gene expression of kiss1 and kiss2
in the brain (4.2 ± 0.2 and 8 ± 0.3-fold change, respectively), the in-
crease of these two neuropeptides was concomitant with an in-
crease in gnrh3 gene expression (7.8 ± 0.3-fold change)
(Gioacchini et al., 2010a).
Concomitantly, the daily numbers of ovulated eggs significantly
increased, the increase was evident from the second day of admin-
istration and remains significantly higher during the 10 days treat-
ment. The higher mean of ovulated eggs x female (81.9 ± 36 and
203.3 ± 59.2 of control and treated fish, respectively) was associ-
ated with a better gamete quality as showed by the highest hatch-
ing rate (65.7 ± 10.8 and 92.2 ± 6.6 of control and treated fish,
respectively) and faster embryonic development (Gioacchini
et al., 2010a). The contribution of probiotic on enhancing follicle
growth phase was supported by the increase of the gonadosomatic
index (GSI) Gioacchini et al., 2011 along with histological studies
on ovary sections, showing an increase of vitellogenic follicles
(Gioacchini et al., 2010a; Gioacchini et al., 2011) in the ovary of
treated fish (Fig. 1).
299
The probiotic treatment also affected the expression of a num-
ber of genes related with the oocyte growth phase including the
cyp19a in the ovary and the vtg and era in the liver as reported
in Table 1 (Gioacchini et al., 2011). Evidence on stimulating roles
of L. rhamnosus on follicle maturation phase is also available (Gio-
acchini et al., 2010a; Gioacchini et al., 2011). An increase of tran-
scription of genes codifying for signals involved on maturation
phase including lhr, 20b-hsd, mprb, cyclin B, activinbA1 and
smad2, concomitant with a down regulation of genes codifying
for local factors able to prevent premature oocyte maturation
including tgfb1, gdf9 and bmp15 were observed as reported in Ta-
ble 1.
Looking at the oocyte protein pattern, changes in cytoplasmic
proteins induced by probiotic treatment were evidenced mainly
during the maturation phase, but also evident during vitellogenesis
(Giorgini et al., 2010). These proteolytic degradation of yolk com-
ponents at higher molecular weight are controlled by cathepsin L
(Carnevali et al., 2006; Carnevali et al., 2008), which shows a sharp
increase in gene expression and enzyme activity (Giorgini et al.,
2010) responsible for the yolk degradation and free amino acids
(FAAs) release.
The macromolecular changes occurring during follicle develop-
ment (Carnevali et al., 2009; Giorgini et al., 2010) evidenced by
Fourier transform infrared (FT-IR) microspectroscopy provided a
biochemical fingerprint of oocytes components, at different devel-
opmental stages (I-II, IIIa, IIIb and IV) and revealed specific vibra-
tional patterns, correlated with the selective up-take of
vitellogenin and with the maturation process. The total absorbance
cartograms and the relative chemical maps evidenced that probi-
otic administration mainly influenced oocytes at the later stages
of development, whose representative spectra showed changes in
the phosphate and glucidic content and in proteins secondary
structures.
Probiotic has effects also on maturational competence acquisi-
tion. Gioacchini et al. (2012) showed that probiotic administration
induces the responsiveness of incompetent follicles (IIIa) to the
maturation inducing hormone (MIH) and their in vitro maturation
(Table 2).
The gene ontology (GO) data analysis correlated several pro-
cesses with L. rhamnosus presence during follicle development
(Gioacchini et al., 2012). The administration of probiotic to mature
females induced changes in class IIIa follicles (maturational incom-
petent) supposedly throughout the control of several genes belong-
ing to biological processes involved in signal transduction and
response to chemical stimuli and regulates transcripts associated
with development (regulation of anatomical structure size) and
biological quality. The GO analysis of molecular function in class
IIIa follicles revealed that probiotic acts on two main groups of
genes: (i) those involved in the selective interaction of a molecule
with one or more specific sites and molecules such as heme bind-
ing, iron ion binding, tetrapyrrole binding, GTP binding and guanyl
ribonucleotide binding; and (ii) those involved in monooxigenase
activity and oxidoreductase activity.
In class IIIb follicles (maturational competent), the GO analysis
evidenced the probiotic ability to influence the transcription of
many genes belonging to biological process and molecular function
including regulation of transcription DNA-dependent, transcrip-
tion factor activity, transcription regulator activity and sequence-
specific DNA binding. In addition, the biological process analysis
evidenced a control of transcripts involved in development includ-
ing multicellular organism development, anatomical structure
development and organ morphogenesis developmental process.
The acquiring of competence of the IIIa follicles was correlated
with an increase of mprb, lhr, activin bA1, and oct4 gene expression
and a decrease of gdf9, tgfb1 and bmp15 (Gioacchini et al., 2012). It
is well known that reproduction is gated by the state of energy re-
300 O. Carnevali et al. / General and Comparative Endocrinology 188 (2013) 297–302

(a)

(b) Regulation Fold Change

GSI Up 1,9±0,1*

vtg Up 63±2,1**
erα Up 4±0,1*

cyp19a Up 2±0,1*

Fig. 1. (a) Representative micrographs of ovaries from (A–C) females fed on L. rhamnosus and (D–F) control females. PV, previtellogenic follicles; V, vitellogenic follicles.
Original magnification A and D 5, B and E 10, C and F 40. (b) Gonadosomatic index (GSI) values fold change of zebrafish females after 10 days L. rhamnosus treatment with
respect to control ones (n = 10). GSI was calculated by expressing the gonad weight as a percentage of the total body weight. The vtg, era in the liver and cyp19a in the ovary
mRNA fold changes, normalized against arp and b-act genes, in zebrafish females fed on L. rhamnosus with respect to control ones (n = 7) are reported. Asterisks denote
significant differences from the control group (P < 0.05) analyzed using Student’s t-test. ⁄(P < 0.05) and ⁄⁄(P < 0.01).

serves in the organism (Hill et al., 2008) and results sensitive to di-
Table 1
verse metabolic cues. In fact, the mechanism through which probi-
lhr, 20bhsd, mpr b, ciclin b, smad 2, activin b, mpra, tgf b1, bmp15, gdf9 fold change
on mRNA levels in the ovary, normalized against b-actin and gapdh genes, in females otics affect oogenesis and fecundity includes several factors. The
fed on L. rhamnosus with respect to control ones (n = 5). Asterisks denote significant neuroendocrine mechanisms accountable for the tight coupling be-
differences from the control group analyzed using Student’s t-test: ⁄(P < 0.05) and tween energy homeostasis and fecundity are represented by neu-
⁄⁄
(P < 0.01).
ropeptides and metabolic hormones, which integrate and exert
Gene name Regulation Fold change an interplay at different levels of the hypothalamus–pituitary–go-
LHR Up 4 ± 0.7⁄ nadal (HPG) axis, governing reproduction (Castellano et al., 2009;
20bHSD Up 3 ± 0.5⁄ Fernandez-Fernandez et al., 2006; Kitahashi et al., 2009; Zohar,
mPR b Up 4 ± 0.3⁄ 2010).
CICLIN B Up 13 ± 1.1⁄⁄ The impact of energy status along the reproductive axis is ex-
SMAD 2 Up 7.5 ± 1.3⁄
ACTIVIN B Up 4.2 ± 0.9⁄
pressed through a number of neuropeptide hormones including
mPRa No variation – kiss1, kiss2, and leptin, peripheral metabolic signals, whose nature
TGF b1 Down 3 ± 0.2⁄ and mechanisms of action became clear only recently in mammals
BMP15 Down 3 ± 0.1⁄ and, to a lesser extent, in fish (Carnevali et al., 2011; Castellano
GDF9 Down 3.2 ± 0.2⁄
et al., 2009; Fernandez-Fernandez et al., 2006; Kitahashi et al.,
2009). Organisms may control the energy availability through lep-
tin levels that induces reproduction by activating kisspeptin neuro-
peptides production at the central nervous system. In mammals,
leptin produced by adipose tissue, also acts at pituitary level stim-
Table 2
Rate (%) of GVBD of zebrafish IIIa and IIIb follicles isolated from control group ovaries
ulating FSH and LH production (Zieba et al., 2005) and at gonadal
incubated in L15 (CTRL) and L15+MIH (MIH) (1 lg/ml, MIH) and of IIIa and IIIb level, controlling the steroidogenesis process (Moschos et al.,
follicles isolated from probiotic group ovaries incubated in L15 (PROBIO) and 2002). Leptin is one of the genes up regulated by probiotic admin-
L15+MIH (PROBIO+MIH), n = 20 bars. Raw data are presented as the mean ± SD. istration in zebrafish intestine and brain; the increase of leptin is
Different letters indicate statistically significant differences (P < 0.05), analyzed using
well correlated with the increase of kisspeptins and gnrh3 levels
ANOVA followed by Bonferroni multiple-comparison test, after arcsine transforma-
tion of raw data. (Gioacchini et al., 2010a).
A recent study investigated the effect of probiotic on autopha-
IIIa IIIb
gic and apoptotic processes in zebrafish preovulatory follicles
a
CTRL 3.00 ± 1 20.74 ± 3b (Gioacchini et al., 2013). The electron microscopy analysis evi-
CTRL+MIH 19.99 ± 2b 69.68 ± 4d
denced higher number of autophagosomes in stage III and IV fol-
PROBIO 6 ± 1.5a 25 ± 2b
PROBIO+MIH 42 ± 3c 95.7 ± 4e licles from females fed the probiotic L. rhamnosus. This result was
in agreement with the higher expression of genes involved in the
autophagic process, i.e. activating molecule in becn-1-regulated
 O. Carnevali et al. / General and Comparative Endocrinology 188 (2013) 297–302
autophagy (ambra1), myosin-like bcl2 interacting protein (becn1),
microtubule-associated protein 1 light chain 3 (lc3) and
UV-irradiation-resistance-associated gene (uvrag) (Gioacchini
et al., 2013). In addition, class III and IV follicles from females
fed the probiotic exhibited more LC3 isoform II (LC3-II) and re-
duced P62 protein (Gioacchini et al., 2013). The increased autoph-
agy in preovulatory follicles from females fed with probiotics was
concomitant with a decrease in the apoptotic process in the ovary,
as evidenced by TUNEL analysis and confirmed by higher expres-
sion of igfII and lower expression of genes involved in apoptosis
[bcl2-associated X protein (bax), apoptotic protease activating fac-
tor-1 (apaf), tumor suppressor protein 53 (p53), and caspasi 3
(cas3)] and igf1r, demonstrating that probiotic can modulate the
balance between apoptosis and autophagy regulating the ovary
physiology improving follicular survival and inhibiting follicular
apoptosis (Gioacchini et al., 2013).
5. Conclusions
Zebrafish has been used in several studies to test whether mic-
robiota composition might influence energy balance and reproduc-
tion. Clear evidence shows that the presence of beneficial microbes
stimulate organism to more efficiently use diet-derived energy
sources, leading to growth improvement. In zebrafish, the treat-
ment with L. rhamnosus from birth to sexual maturation seems
to influence larval development by accelerating growth and back-
bone calcification and anticipating sex differentiation. When
administered to adults, L. rhamnosus influences the maturational
competence and improves the number of vitellogenic follicles
and GSI, both related with the higher number of ovulated eggs.
Finally, changes in leptin’s expression are also recorded, to-
gether with an increase of kisspeptins and gnrh3 levels supporting
the positive role of probiotic on D. rerio reproduction.
Acknowledgments
This paper is dedicated to our colleague and dear friend Maria
Fosca Franzoni that died prematurely, to remember her great con-
tributions to comparative endocrinology, confident that her legacy
continue to impact and shape this field.
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