Artificial
Neural Network (ANN) as a Tool to Reduce Human-Animal Interaction Improves
Senegalese Sole Production
ABSTRACT
Manipulation is usually required for biomass calculation and food estimation for optimal fish growth in production facilities. However, the advances in computer-based systems have opened a new range of applied possibilities. In this study we used image analysis and a neural network algorithm that allowed us to successfully provide highly accurate biomass data. This developed system allowed us to compare the effects of reduced levels of human-animal interaction on the culture of adult Senegalese sole (Solea senegalensis) in terms of body weight gain. For this purpose, 30 adult fish were split into two homogeneous groups formed by three replicates (n=5) each: a control group (CTRL), which was standard manipulated and an experimental group (EXP), which was maintained under a lower human-animal interaction culture using our system for biomass calculation. Visible implant elastomer was, for the first time, applied as tagging technology for tracking soles during the experiment (four months). The experimental group achieved a statistically significant weight gain (p<0.0100) while CTRL animals did not report a statistical before-after weight increase. Individual body weight increment was lower (p<0.0100) in standard-handled animals. In conclusion, our experimental approach provides evidence that our developed system for biomass calculation, which implies lower human-animal interaction, improves biomass gain in Senegalese sole individuals in a short period of time.
Long Exposure to a Diet Supplemented with Antioxidant and Anti-Inflammatory Probiotics Improves Sperm Quality and Progeny Survival in the Zebrafish Model
David G. Valcarce, Marta F. Riesco, Juan M. Martínez-Vázquez and Vanesa Robles
Biomolecules, 2019
ABSTRACT
Effect of low sperm quality on progeny: a study on zebrafish as model species
Marta F. Riesco, David G. Valcarce, Juan M. Martínez-Vázquez and Vanesa Robles
ABSTRACT
The Use of Antifreeze Proteins in the Cryopreservation of Gametes and
Embryos
ABSTRACT
Non-coding RNA regulation in reproduction: Their potential use as
biomarkers
ABSTRACT
Diet Supplemented with Antioxidant and Anti-Inflammatory Probiotics
Improves Sperm Quality after Only One Spermatogenic Cycle in Zebrafish Model
ABSTRACT
Solea senegalensis male reproductive dysfunction: new insights into
an unsolved question
ABSTRACT
Evaluation of Intracellular Location of Reactive Oxygen Species in Solea
senegalensis spermatozoa
ABSTRACT
Effect of captivity and cryopreservation on ROS production in Solea
senegalensis spermatozoa
ABSTRACT
Epigenetics in fish gametes and early embryo
ABSTRACT
Molecular basis of spermatogenesis and sperm quality
ABSTRACT
Selection of non-apoptotic sperm by magnetic activated cell sorting in
Senegalese sole (Solea senegalensis)
ABSTRACT
Paternal contribution to development: Sperm genetic damage and repair in
fish
ABSTRACT
Considering the
implications that the integrity of sperm genome and epigenome has on the
preservation of a proper genotype and phenotype in the progeny, the methods
applied for the study of chromatin damage and for the study of transcriptome
are described. Moreover we discuss some injuring agents affecting paternal
information, from the presence of contaminants in the aquatic environment, to
the reproductive practices applied in fish farming. The consequences of
fertilizing with damaged spermatozoa, as well as the zygotic ability
to repair damage are also reviewed.
Artificial
Neural Network (ANN) as a Tool to Reduce Human-Animal Interaction Improves
Senegalese Sole Production
Juan M. Martínez-Vázquez, David G. Valcarce, Marta F. Riesco,
Vicent Sanz-Marco, Morito Matsuoka, Vanesa Robles
Biomolecules, 2019
ABSTRACT
ABSTRACT
Manipulation is usually required for biomass calculation and food estimation for optimal fish growth in production facilities. However, the advances in computer-based systems have opened a new range of applied possibilities. In this study we used image analysis and a neural network algorithm that allowed us to successfully provide highly accurate biomass data. This developed system allowed us to compare the effects of reduced levels of human-animal interaction on the culture of adult Senegalese sole (Solea senegalensis) in terms of body weight gain. For this purpose, 30 adult fish were split into two homogeneous groups formed by three replicates (n=5) each: a control group (CTRL), which was standard manipulated and an experimental group (EXP), which was maintained under a lower human-animal interaction culture using our system for biomass calculation. Visible implant elastomer was, for the first time, applied as tagging technology for tracking soles during the experiment (four months). The experimental group achieved a statistically significant weight gain (p<0.0100) while CTRL animals did not report a statistical before-after weight increase. Individual body weight increment was lower (p<0.0100) in standard-handled animals. In conclusion, our experimental approach provides evidence that our developed system for biomass calculation, which implies lower human-animal interaction, improves biomass gain in Senegalese sole individuals in a short period of time.
Long Exposure to a Diet Supplemented with Antioxidant and Anti-Inflammatory Probiotics Improves Sperm Quality and Progeny Survival in the Zebrafish Model
David G. Valcarce, Marta F. Riesco, Juan M. Martínez-Vázquez and Vanesa Robles
Biomolecules, 2019
ABSTRACT
The aim of the present experiment is to study the effects of oral ingestion of a mixture of two probiotic bacteria on sperm quality and progenies. Three homogeneous groups of juvenile zebrafish were created. Once having reached adulthood (3 months postfertilization; mpf), each group received different feeding regimens: a standard diet (control), a maltodextrin-supplemented diet (vehicle control), or a probiotic-supplemented diet (a mixture (1:1) of Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347). The feeding regime lasted 4.5 months. Growth parameters (weight and length) were determined at 3, 5, and 7.5 mpf. Sperm motility was evaluated using computer-assisted sperm analysis at 5 and 7.5 mpf. Progeny survival, hatching rate, and malformation rate were also evaluated. Results showed that probiotic-supplemented diet improved growth parameters compared with the standard diet. The highest percentage of motile spermatozoa was reported in the probiotic-fed group. Concomitantly, the percentage of fast sperm subpopulation was significantly lower in samples derived from control males. Furthermore, there was a significant difference in progeny survival between the probiotic-fed group and the control group at three developmental times (24 hours postfertilization (hpf), 5 days postfertilization (dpf) and 7 dpf). In conclusion, in zebrafish, prolonged ingestion of a mixture of Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347 has positive effects on growth, sperm quality, and progeny survival.
Effect of low sperm quality on progeny: a study on zebrafish as model species
Marta F. Riesco, David G. Valcarce, Juan M. Martínez-Vázquez and Vanesa Robles
Scientific Reports, 2019
ABSTRACT
Nowadays a decrease tendency in human sperm quality has been reported mainly in developed countries. Reproductive technologies have been very valuable in achieving successful pregnancies with low quality sperm samples. However, considering that spermatozoa molecular contribution is increasingly important in recent studies, it is crucial to study whether fertilization with low sperm quality could leave a molecular mark on progeny. This study explores the consequences that fertilization with low sperm quality may have on progeny, using zebrafish as a model. Good and bad breeders were established attending to sperm quality analyses and were individually tracked. Significant differences in fertilization and malformation rates were obtained in progenies between high and low quality sperm samples. Moreover an altered miR profile was found in the progenies of bad zebrafish breeders (upregulation of miR-141 and miR -122 in 24 hpf embryos) and as a consequence, some of their targets involved in male sex development such as dmrt1, suffered downregulation. Our results indicate that fertilizing with high sperm quality samples becomes relevant from a new perspective: to avoid molecular alterations in the progeny that could remain masked and therefore produce unexpected consequences in it.
The Use of Antifreeze Proteins in the Cryopreservation of Gametes and Embryos
Vanesa Robles, Marta F. Riesco and David
G. Valcarce.
Biomolecules, 2019
ABSTRACT
The
cryopreservation of gametes and embryos is a technique widely used in
reproductive biology. This technology helps in the reproductive management of
domesticated animals, and it is an important tool for gene banking and for
human-assisted reproductive technologies. Antifreeze proteins are naturally
present in several organisms exposed to subzero temperatures. The ability for
these proteins to inhibit ice recrystallization together with their ability to
interact with biological membranes makes them interesting molecules to be used
in cryopreservation protocols. This mini-review provides a general overview
about the use of antifreeze proteins to improve the short and long term storage
of gametes and embryos.
Non-coding RNA regulation in reproduction: Their potential use as
biomarkers
Vanesa Robles, David G.
Valcarce, Marta F. Riesco.
Non-coding RNA Research, 2019
ABSTRACT
Non-coding RNAs
(ncRNAs) are crucial regulatory elements in most biological processes and
reproduction is also controlled by them. The different types of ncRNAs, as well
as the high complexity of these regulatory pathways, present a complex
scenario; however, recent studies have shed some light on these questions,
discovering the regulatory function of specific ncRNAs on concrete reproductive
biology processes. This mini review will focus on the role of ncRNAs in
spermatogenesis and oogenesis, and their potential use as biomarkers for
reproductive diseases or for reproduction success.
Diet Supplemented with Antioxidant and Anti-Inflammatory Probiotics
Improves Sperm Quality after Only One Spermatogenic Cycle in Zebrafish Model
David G. Valcarce, Marta F. Riesco,
Juan Manuel Martínez-Vázquez, Vanesa Robles.
Nutrients, 2019
ABSTRACT
Infertility is a
medical concern worldwide and could also have economic consequences in farmed
animals. Developing an efficient diet supplement with immediate effects on
sperm quality is a promising tool for human reproduction and for domesticated
animal species. This study aims at elucidating the effect of a short-time
probiotic supplementation consisting of a mixture of two probiotic bacteria
with proven antioxidant and anti-inflammatory activities on zebrafish sperm
quality and male behavior. For this purpose, three homogeneous groups of males
in terms of motility (<60%) were established. The control group was fed with
a normal standard diet. The other received supplements: One group (vehicle
control) was fed with maltodextrin and the other received a probiotic
preparation based on a mixture (1:1) of Lactobacillus rhamnosus CECT8361
and Bifidobacterium longum CECT7347. The feeding regime was 21
days corresponding with a single spermatogenesis in zebrafish. The preparation
did not modify animal weight, positively affected the number of fluent males,
increased sperm concentration, total motility, progressive motility, and fast
spermatozoa subpopulations. Moreover, the animals fed with the supplement
showed different behavior patterns compared to control groups. Our results
suggest a diet-related modulation on the exploration activity indicating a
lower stress-like conduct. The studied formulation described here should be
considered as advantageous in male reproductive biotechnology.
Solea senegalensis male reproductive dysfunction: new insights into
an unsolved question
Marta F. Riesco, David G. Valcarce, Juan
Manuel Martínez-Vázquez, Ignacio Martín, Andrés Calderón-García, Verónica
González-Núñez, Vanesa Robles.
Reproduction, Fertility and Development, 2019
ABSTRACT
Senegalese sole (Solea
senegalensis) is a species with a high commercial value that exhibits a
reproductive dysfunction in males born and raised in captivity (F1)
that hinders their sustainable culture. The present study evaluates the sperm
quality and dopaminergic pathway of males born in the wild environment and of F1 males.
Traditional sperm analyses were performed, finding only significant differences
in curvilinear velocity (VCL) and no significant differences in viability and
total motility. No differences in global sperm methylation were observed either
in spermatozoa or brain between the two groups (F1 and
wild-born males). However, our results point to a different sperm molecular
signature between wild fish and fish born in captivity, specifically the
differential expression in miR-let7-d and miR-200a-5p between these two groups.
miR-let7-d has been correlated with spermatogenesis and sex
preferences, whereas the miR-200 family is implied in target innervation of
dopaminergic neurons in zebrafish. When we analysed the dopaminergic pathway,
no differences were found in terms of different mRNA expression of dopaminergic
markers. However, some differences were detected in terms of tyrosine
hydroxylase protein expression by western blot analysis, thus suggesting an
altered post-transcriptional regulation in F1 males. The
results of this study suggest that an altered sperm miRNA signature in F1 males
could be one possible mode of transmission of reproductive dysfunction to the
progeny.
Evaluation of Intracellular Location of Reactive Oxygen Species in Solea
senegalensis spermatozoa
David G. Valcarce
and Vanesa Robles
JoVE, 2018
ABSTRACT
Oxidative stress
is one of the important factors in decreasing sperm quality. Developing
efficient protocols for detecting reactive oxygen species (ROS) in spermatozoa
is of high importance in any species, but these methods are rarely used and
even less in teleost. Cryopreservation is a useful technique in aquaculture for
different purposes, including gene banking and guaranteed sperm availability
throughout the year. Freezing/thawing procedures could cause ROS production and
damage the sperm cells. Considering the prospective damage that an excess of
ROS production could cause in spermatozoa depending on their localization, here
a detailed methodology to detect H2O2 and to evaluate its intracellular
localization by confocal microscopy is provided. For this purpose, a
combination of 3 fluorochromes (2′,7′-Dichlorodihydrofluorescein diacetate
(DCFH-DA), a live mitochondria stain and 4′,6-Diamidino-2-phenylindole
dihydrochloride (DAPI)) are used to evaluate the co-localization of
H2O2 with spermatozoa nuclei or mitochondria in Solea senegalesis sperm
samples.
Effect of captivity and cryopreservation on ROS production in Solea
senegalensis spermatozoa
David G. Valcarce
and Vanesa Robles
Reproduction, 2016
ABSTRACT
Reactive oxygen
species have a great impact on spermatozoa function. Gametes from sole males
born in captivity (F1) display lower quality than those from wild individuals.
In this paper the percentage of positive cells for dichlorofluorescein (DCF+)
was determined by flow cytometry in wild and F1 animals, the effect of
cryopreservation on DCF+ cells was evaluated in both groups and the
distribution of H2O2 within the cell was studied by confocal microscopy. Our
results indicated that there are no differences in either viability or DCF+
cells between wild and F1 animals when fresh samples were evaluated. However,
when data were analyzed considering two different sperm populations in terms of
motility, a significant decrease in viability and DCF+ cells was reported in
low motile F1 spermatozoa. Cryopreservation did not alter viability or the
presence of DCF+ cells in sperm samples from wild animals, but significantly
decreased viability in F1 samples. Distribution patterns of H2O2 have been
established by confocal microscopy in Solea senegalensis spermatozoa:
colocalization of H2O2 with active mitochondria (MitoTracker+) and
colocalization with nuclear DNA (DAPI). When compared with H2O2 distribution in
other marine species such as Scophthalmus maximus, Solea senegalensis spermatozoa
showed widespread presence of H2O2 particularly in the nuclei, which could
potentially compromise DNA integrity.
Epigenetics in fish gametes and early embryo
Catherine Labbé, Vanesa Robles, María Paz
Herráez
Aquaculture, 2017
ABSTRACT
The spermatozoa
and oocyte are very specialized cells which possess the unique ability to fuse
and to produce an embryo. This embryo will develop into a mature organism
capable of producing gametes again. This colloquial statement comprehends
however the most complex mechanisms in biology that is cell differentiation and
cell reprogramming. Epigenetic modifications are major actors in this process,
both during gametogenesis and embryo development. The epigenetic concept is
resulting from the growing scientific understanding of embryology and genetics,
and it is made operational by generic molecular mechanisms, namely DNA
methylation, histone tails modifications, and non-coding RNAs. Epigenetic
modifications associated with gamete differentiation and early embryo
development are quite well described in mammals, but increasing research is
developed in fish today, either in model or in aquaculture species. Because
fish are living organisms thoroughly exposed to temperature and to water
quality, environment is likely to influence the epigenetic pattern of the
gametes, embryo, or adult organisms. Reproductive biotechnologies are also at
risk to modify gametes and embryo epigenetics. This review is aimed towards
giving the basic knowledge on the molecular actors of epigenetics, highlighting
the developing knowledge in mammals which can be useful to fish research and
proposing the possible area where epigenetics can enrich research on fish
gametes and reproductive technologies.
Molecular basis of spermatogenesis and sperm quality
Vanesa Robles, M. Paz Herráez, Catherine
Labbé, Elsa Cabrita, Martin Pšenicka, David G. Valcarce, Marta F. Riesco
General and Comparative Endocrinology, 2017
ABSTRACT
Spermatozoan
quality can be evaluated in different ways, here we focus on the analysis of
DNA, RNA and epigenetic status of germ cells. These characterizations also can
be the bases for explaining sperm quality at other levels, so we will see how
some of these molecules could affect other sperm quality markers. Moreover, we
consider the possibility of using some of these molecules as predictors of
sperm quality in terms of the ability to produce healthy offspring. The
relevant effect of different types of RNA molecules in germ line specification
and spermatogenesis and the importance of germ cell DNA integrity and a proper
epigenetic pattern will be also discussed. Although most studies at this level
have been performed in mammals, some information is available for fish; these
recent discoveries in fish models are included. We provide a general overview
on how these molecules could have a deep influence in the final sperm quality.
Selection of non-apoptotic sperm by magnetic activated cell sorting in
Senegalese sole (Solea senegalensis)
David G. Valcarce, M. Paz Herráez, O.
Chereguini, C. Rodríguez, V. Robles
Theriogenology, 2016
ABSTRACT
Senegalese sole (Solea
senegalensis) is a promising species in aquaculture. However, due to
decreased sperm quality in F1 generations and the absence of courtship in those
individuals born in captivity, artificial fertilization is being used to
generate new progenies. The objective of this study was to implement a sperm
selection method for non-apoptotic sperm subpopulation recovery prior to sperm
cryopreservation. In particular, magnetic activated cell sorting (MACS) is used
to eliminate apoptotic spermatozoa. This study represents the proof-of-concept
for MACS applicability in teleost species relevant in aquaculture. Apoptotic
cell population was studied by flow cytometry using YO-PRO 1 and a caspase
detection kit. Also, reactive oxygen species were measured in sperm samples.
Our data demonstrated that caspase detection is more specific than YO-PRO 1 in
the identification of apoptotic cells in S. senegalensis seminal
samples. The results showed that the percentage of apoptotic cells
(caspase positive) was significantly higher (p=0.04) in seminal samples from F1
than from wild individuals. MACS removed a significant number of apoptotic
cells from the samples (54% and 75% in wild and F1 individuals, respectively),
decreasing the level of cells positive for reactive oxygen species (p=0.17). In
conclusion, this technique reduces the percentage of non-functional spermatozoa
in a seminal sample prior to cryopreservation. This novel technique can be
applied directly in the aquaculture industry.
Paternal contribution to development: Sperm genetic damage and repair in
fish
M.P. Herráez, J. Ausió, A. Devaux, S.
González-Rojo, C. Fernández-Díez, S. Bony, N. Saperas, V. Robles
Aquaculture, 2017
ABSTRACT
In this review we
provide an overview of the components of the spermatozoa playing an
important role in reproductive success beyond fertilization, showing
the relationship between the integrity of the diverse elements and the development
of a healthy offspring. The present knowledge about fish sperm chromatin
organization, epigenetic modifications of DNA and histones and
sperm-borne RNAs, essential in controlling embryo development, is summarized,
pointing out the possibility of using specific genes or transcripts as
biomarkers of sperm quality. Data about commercial species are reported
when available and more detailed information about zebrafish sperm is presented.
Considering the
implications that the integrity of sperm genome and epigenome has on the
preservation of a proper genotype and phenotype in the progeny, the methods
applied for the study of chromatin damage and for the study of transcriptome
are described. Moreover we discuss some injuring agents affecting paternal
information, from the presence of contaminants in the aquatic environment, to
the reproductive practices applied in fish farming. The consequences of
fertilizing with damaged spermatozoa, as well as the zygotic ability
to repair damage are also reviewed.