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With increasing evidence showing the connections between the microbiome, neurophysiology, and behavior, our research endeavors to investigate whether the consumption of a combination of two postbiotics with antioxidant effects can affect behavior regulation in model species. Here, we worked with a combination (1:1 ratio) of heat-treated Bifidobacterium longum subsp. longum ES1 (CECT7347) and Lacticaseibacillus rhamnosus BPL15 (CECT8361) as a dietary supplement. To examine the potential benefit of using this formulation to alleviate anxiety-like behavior, we employed two model species, Caenorhabditis elegans and adult Danio rerio . In C. elegans , the postbiotic supplementation reduced the anxiety-related behavior analyzed by means of the octanol avoidance test. In zebrafish, the novel tank test indicated a different swimming pattern 2 and 4 months after the animals were fed with the postbiotic combination. While fish did not exhibit any variance in their locomotion parameters such as pace and speed, they showed a statistically significant preference to spend more time in the upper zone of the water tank, a behavior that is correlated with a lower anxiety-like behavior in these species. Our aim with this study is to present evidence that can be used to develop whole-cell postbiotic-based novel and innovative dietary supplements for anxiety-related conditions.

Aquaculture routine practices may cause stress induction on the fish and compromise their welfare affecting the production. This experiment aimed to evaluate the potential links between handling during culture with stress responses and growth on Senegalese sole (Solea senegalensis). We worked with two fish cohorts in terms of initial body weight and culture stage: Trial 1 included specimens in the fattening stage (226 ± 4.96 g) and Trial 2 animals in the pre-fattening stage (27.20 ± 0.44 g). The tested culture protocol, which lasted 6 and 4 months for Trial 1 and 2, respectively, mainly reduced handling-derived stressors in the experimental tanks via lowering routine samplings to a minimum. This decrease of the handling-derived stress was reflected in both trials with lower concentration of circulating cortisol in blood plasma from the experimental fish when compared to controls. Moreover, the proposed protocol promoted higher growth in the fish cultured in the less disturbing protocol in Trial 2. Higher specific growth rates and mean body weight and length were reported. In order to further explore the potential beneficial effects of our protocol, we studied the musculoskeletal from Trial 2 gene expression of key genes regulating glucocorticoid signaling pathway and apoptosis: glucocorticoid receptors 1 and 2 (gr1, gr2), heat shock protein 90 AA (hsp90aa), and caspase 6 (casp6). In line with the cortisol reduced level in this trial, gr1, hsp90aa, and casp6 genes showed lower expression in the samples coming from the experimental group. The findings of this study provide valuable information to the aquaculture industry for the management of Solea senegalensis stress and welfare., Competing Interests: Declarations Ethical approval All experimental procedures on fish were authorized by the Bioethics and Animal Welfare Committees of the facilities under the project registration number ULE009-2020 and were conducted following the Spanish and European regulations for the use of laboratory animals. Competing interests The authors declare no competing interests., (© 2023. The Author(s).)

The effects of stress during early vertebrate development can be especially harmful. Avoiding stressors in fish larvae is essential to ensure the health of adult fish and their reproductive performance and overall production. We examined the consequences of direct exposure to successive acute stressors during early development, including their effects on miR-29a and its targets, survival, hatching and malformation rates, larval behaviour and cartilage and eye development. Our aim was to shed light on the pleiotropic effects of early-induced stress in this vertebrate model species. Our results showed that direct exposure to successive acute stressors during early development significantly upregulated miR-29a and downregulated essential collagen transcripts col2a1a, col6a2 and col11a1a, decreased survival and increased malformation rates (swim bladder, otoliths, cardiac oedema and ocular malformations), promoting higher rates of immobility in larvae. Our results revealed that stress in early stages can induce different eye tissular architecture and cranioencephalic cartilage development alterations. Our research contributes to the understanding of the impact of stressful conditions during the early stages of zebrafish development, serving as a valuable model for vertebrate research. This holds paramount significance in the fields of developmental biology and aquaculture and also highlights miR-29a as a potential molecular marker for assessing novel larval rearing programmes in teleost species., (© 2024. The Author(s).)

Recent research has provided compelling evidence demonstrating that paternal exposure to different stressors can influence their offspring's phenotypes. We hypothesized that paternal stress can negatively impact the progeny, altering different miRs and triggering different physiological alterations that could compromise offspring development. To investigate this, we exposed zebrafish male siblings to a chronic stress protocol for 21 days. We performed RNA-sequencing (RNA-seq) analyses to identify differentially expressed small noncoding RNAs in 7-day postfertilization (dpf) larvae derived from paternally stressed males crossed with control females compared with the control progeny. We found a single miRNA differentially expressed-miR-29a-which was validated in larva and was also tested in the sperm, testicles, and brain of the stressed progenitors. We observed a vertical transmission of chronic stress to the unexposed larvae, reporting novel consequences of paternally inherited chronic stress at a molecular level. The deregulation of mi-R29a in those larvae could affect relevant biological processes affecting development, morphogenesis, or neurogenesis, among others. Additionally, these disruptions were associated with reduced rates of survival and hatching in the affected offspring.

Background: Chronic stress can produce a severe negative impact on health not only in the exposed individuals but also in their offspring. Indeed, chronic stress may be contributing to the current worldwide scenario of increasing infertility and decreasing gamete quality in human populations. Here, we evaluate the effect of chronic stress on behavior and male reproductive parameters in zebrafish. Our goal is to provide information on the impact that chronic stress has at molecular, histological, and physiological level in a vertebrate model species., Results: We evaluated the effects of a 21-day chronic stress protocol covering around three full waves of spermatogenesis in Danio rerio adult males. The induction of chronic stress produced anxiety-like behavior in stressed males as assessed by a novel tank test. At a molecular level, the induction of chronic stress consistently resulted in the overexpression of two genes related to endoplasmic reticulum (ER) stress in the brain. Gene set enrichment analysis (GSEA) of testes suggested a dysregulation of the nonsense-mediated decay (NMD) pathway, which was also confirmed on qPCR analysis. Histological analysis of the testicle did not show significant differences in terms of the relative proportions of each germ-cell type; however, the quality of sperm from stressed males was compromised in terms of motility. RNA-seq analysis in stress-derived larval progenies revealed molecular alterations, including those predicted to affect translation initiation, DNA repair, cell cycle control, and response to stress., Conclusions: Induction of chronic stress during a few cycles of spermatogenesis in the vertebrate zebrafish model affects behavior, gonadal gene expression, final gamete quality, and progeny. The NMD surveillance pathway (a key cellular mechanism that regulates the stability of both normal and mutant transcripts) is severely affected in the testes by chronic stress and therefore the control and regulation of RNAs during spermatogenesis may be affected altering the molecular status in the progeny., (© 2023. The Author(s).)

Reactive oxygen species (ROS) could have a negative impact on sperm cellular function and viability. This chapter describes a protocol for oxidative stress evaluation using dichlorofluorescein (DCF) which can specifically reveal intracellular reactive oxygen species. The protocol described here has been used in human and teleost species sperm samples. The method can be used with two approaches: (1) flow cytometry, for quantification of DCF + cells, or (2) confocal microscopy, for the localization of ROS within the cells.

Primordial germ cells (PGCs) are unique cells in an embryo. These cells contain all genetic information and therefore represent the best source to store maternal and paternal genomes until embryo cryopreservation is achieved. However, the number of these cells in an embryo is very low limiting their potential application in cryopreservation and surrogate production. However, it was assumed that the induction of fish PGCs in vitro is not possible because in vivo they inherit germ plasm. In this chapter, we describe a successful differentiation protocol explaining the crucial factors and steps for in vitro PGC generation.

There is increasing evidence that gut microbiome could have effects on neurological processes and on behavior. In this study we used the novel tank test (NTT) to analyze zebrafish exploring behavior after four months' supplementation with probiotics with probed antioxidant and anti-inflammatory properties. Results showed that prolonged ingestion of Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347 significantly alters the swimming pattern and mean swimming speed in the zebrafish model. After treatment, zebrafish strongly reduced their bottom-dwelling geotactic behavior when placed in a new tank, which could be correlated to a lower state of anxiety., (© 2020 Published by Elsevier Ltd.)

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.

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.

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 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., Competing Interests: The authors declare no conflict of interest.

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.

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.

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.

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.

The synthetic estrogen 17-α-ethinylestradiol (EE2), a major constituent in contraceptive pills, is an endocrine disrupting chemical (EDC) present in the aquatic environment at concentrations of ng/L. Developmental exposure to these low concentrations in fish can induce several disorders. Zebrafish (Danio rerio) is a perfect organism for monitoring the effects of environmental contaminants. Our hypothesis is that changes promoted by EE2 in the germ line of male adults could be transmitted to the unexposed progeny. We exposed male zebrafish to 2.5, 5 and 10ng/L of EE2 during spermatogenesis and mated them with untreated females. Detailed progeny development was studied concentrating to survival, hatching and malformations. Due to the high incidence of lymphedemas within larvae, we performed qPCR analysis of genes involved in lymphatic development (vegfc and vegfr3) and endothelial cell migration guidance (cxcr4a and cxcl12b). Estrogen receptor (ER) transcript presence was also evaluated in sperm, testis and embryos. Progenies showed a range of disorders although at a low incidence: skeletal distortions, uninflated swimbladder, lymphedema formation, cartilage deformities and otolith tethering. Swimming evaluation revealed less active locomotion. All these processes are related to pathways involving ERs (esr1, esr2a and esr2b). mRNA analysis revealed that environmental EE2 causes the up-regulation of esr1 an esr2b in testis and the increase of esr2b transcripts in sperm pointing to a link between lymphedema in embryos and ER expression impairment. We demonstrate that the effects induced by environmental toxicants can be paternally inherited and point to the changes on the sperm transcriptome as the responsible mechanism., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The objective of this study is to analyse the effect of the ingestion of two selected antioxidant probiotics strains (Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347) on sperm quality parameters in asthenozoospermic males after three and six weeks of administration. Nine asthenozoospermic men without any medical treatment under similar diet conditions participated in the study. The quality of individual sperm samples was evaluated before (previous to ingestion), during (after 3 and 6 weeks of ingestion) and after probiotic administration (3 and 6 weeks after finishing the treatment). Sperm motility was evaluated by computer-assisted sperm analysis system, DNA fragmentation by sperm chromatin structure assay, cell viability by flow cytometry and measurement of intracellular H 2 O 2 (reactive oxygen species; ROS) by flow cytometry using dichloro-dihydrofluorescein diacetate. Sperm motility was drastically improved after the treatment (approximately 6 fold change), DNA fragmentation was statistically reduced after probiotic administration from (approximately 1.2 fold change) and intracellular H 2 O 2 level was decreased (approximately 3.5 fold change). Cell viability was not affected by the treatment. The significant improvement in sperm motility and the decrease in DNA fragmentation reported in this study provide preliminary evidence that probiotics could be administrated to improve motility and decrease DNA fragmentation and ROS levels in asthenozoospermic human males.

Cryopreservation is a well-established technique commonly used in clinical practice. It is used widely for the conservation of gametes and embryos that will be used later for insemination or in vitro fertilization. However, several studies have shown that this technique can produce changes in messenger RNA levels, in the epigenome and induce DNA damage. Although the embryo has potent mechanisms for DNA repair, and molecular changes in spermatozoa are not necessarily reflected in the embryo, it is important to explore new molecular tests and diagnostic tools to design optimal cryopreservation protocols and avoid undesirable molecular alterations. This chapter describes a protocol to quantify the lesions produced by cryopreservation using a protocol previously published by Rothfuss.

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 cells positive 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 the viability or the presence of DCF(+) cells in sperm samples from wild animals, but significantly decreased the viability in F1 samples. Distribution patterns of H2O2 have been established by confocal microscopy in Solea senegalensis spermatozoa: co-localization of H2O2 with active mitochondria (MitoTracker(+)) and co-localization with nuclear DNA (DAPI). 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., (© 2016 Society for Reproduction and Fertility.)

Senegalese sole (Solea senegalensis) is a promising species in aquaculture. However, owing 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 nonapoptotic sperm subpopulation recovery before sperm cryopreservation. In particular, magnetic-activated cell sorting is used to eliminate apoptotic spermatozoa. This study represents the proof-of-concept for magnetic-activated cell sorting 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 that from wild individuals. Magnetic-activated cell sorting 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 nonfunctional spermatozoa in a seminal sample before cryopreservation. This novel technique can be applied directly in the aquaculture industry., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The possibility of generating primordial germ cells (PGCs) in vitro from noncommitted embryonic cells represents an extremely useful tool in current research. Primordial germ cell in vitro differentiation has been successfully reported in mammals. However, contrary to fish, PGC specification in mammals is an inductive mechanism. This study is the first to date to describe a rapid method for PGC in vitro differentiation in teleosts. Primordial germ cell-like cells were characterized by several lines of evidence, including gene expression, cell complexity, size, and image analysis for the quantification of fluorescence under vasa promoter. Moreover, differentiated cells were able to colonize the genital ridge after transplantation. Differentiation treatments increased the number of PGCs in culture, causing differentiation of cells rather than inducing their proliferation. These results open up the possibility of differentiating genetically modified embryonic cells to PGC-like cells to ensure their transmission to the progeny and could be crucial for an in-depth understanding of germline differentiation in teleosts., (© 2014 by the Society for the Study of Reproduction, Inc.)

Sperm cryopreservation is widely used in clinic for insemination, in vitro fertilization and other procedures such as intracytoplasmic sperm injection. The assessment after freezing/thawing of spermatozoa viability, motility and sometimes DNA integrity (mainly using fragmentation assays) has been considered enough to guarantee the safety and effectiveness of the technique. However, it is known that, even when fragmentation is absent, a significant DNA damage could be detected in some genome regions. This is particularly important considering that, during the last years, several studies have pointed out the importance of key paternal genes in early embryo development. In this study, using normozoospermic donors, we present a candidate gene approach in which we quantify the number of lesions produced by freezing/thawing over key genes (PRM1, BIK, FSHB, PEG1/MEST, ADD1, ARNT, UBE3A, SNORD116/PWSAS) using quantitative PCR. Our results demonstrated that the cryopreservation protocol used, which is routinely employed in clinic, produced DNA lesions. The genes studied are differentially affected by the process, and genome regions related to Prader-Willi and Angelman syndromes were among the most damaged: SNORD116/PWSAS (4.56 ± 1.84 lesions/10 kb) and UBE3A (2.22 ± 1.3 lesions/10 kb). To check if vitrification protocols could reduce these lesions, another experiment was carried out studying some of those genes with higher differences in the first study (FSHB, ADD1, ARNT and SNORD116/PWSAS). The number of lesions was not significantly reduced compared to cryopreservation. These results could be relevant for the selection of the most adequate available cryopreservation protocol in terms of the number of lesions that produced over key genes, when no differences with other traditional techniques for DNA assessment could be detected., (© 2013 American Society of Andrology and European Academy of Andrology.)

During recent years, several studies have pointed out the importance of key paternal transcripts in early embryo development. Sperm cryopreservation is commonly applied in assisted reproductive technologies (ARTs) and it is important to know if it produces any relevant effect at this level. In this study, using normozoospermic donors, we present a candidate transcript approach in which we quantify the presence of sperm mRNAs considered as markers for male fertility and pregnancy success. Analyses were done using quantitative PCR. Our results demonstrated that the used cryopreservation protocol, which is routinely employed in clinical practice, alter transcripts considered as spermatozoa quality markers and markers for pregnancy success. Most of the studied transcripts considered as male quality markers (PRM1, PRM2, and PEG1/MEST) and one of studied mRNAs reported as markers of pregnancy success (ADD1) were reduced after cryopreservation. In order to check if vitrification protocols could reduce this alteration, another assay was carried out analyzing those transcripts with higher differences in the first study (PRM1 and PRM2). The results showed the same tendency. Although maternal mRNAs can compensate these deficiencies, these results could make advisable the optimization of freezing/thawing procedures., (Copyright © 2013 Elsevier Inc. All rights reserved.)