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Cardiovascular diseases (CVDs) and diabetes are the primary causes of death in the Philippines. This manuscript reviewed previous studies on the use of predictive analytics for CVD and diabetes risk scoring. This paper also discussed barriers and strategies on how to access/generate available data sets for CVDs and diabetes in the country. CVD and diabetes risk scoring requires the availability of data sets related to such diseases. Although the Philippines has taken strides to implement the Philippine National eHealth solution, such a program does not include strategies toward the use of predictive analytics for CVD and diabetes risk scoring. CVD and diabetes risk scoring research is particularly limited in the Philippines due to challenges related to costs, gaps in policies, and stakeholder involvement. A possible theoretical framework for the analysis and utilization of data sets as well as recommendations and research directions were discussed in this manuscript.

The present study was conducted to investigate the effects of dietary fish oil replacement with a mixture of vegetable oils and probiotic supplementation on plasma biochemical parameters, oxidative stress, and antioxidant ability of Seriola dumerili. Specimens with an initial weight of 175 g were used. Four feeds were formulated with 0% (FO-100), 75% (FO-25), and 100% (FO-0 and FO-0+ with the addition of Lactobacillus probiotics) substitution of fish oil with a mixture of linseed, sunflower, and palm oils. After 109 days, no significant differences were observed in the activity of antioxidant enzymes in the liver, foregut, and hindgut, only glucose-6-phosphate dehydrogenase activity in the liver was higher in the fish fed the FO-100 diet than in those fed the FO-0 diet. No significant differences were observed in the total, reduced, and oxidized glutathione and the oxidative stress index in the liver. In addition, lipid peroxidation in the liver and red muscle values were higher in the fish fed the FO-100 diet than in the fish fed the FO-0+ diet, however, the foregut of the fish fed the FO-100 diet presented lower values than that of the fish fed the FO replacement diet, with and without probiotics. There were significant differences in cholesterol levels in the FO-100 group; they were significantly higher than those observed with the fish diets without fish oil. To sum up, fish oil can be replaced by up to 25% with vegetable oils in diets for Seriola dumerili juveniles, but total fish oil substitution is not feasible because it causes poor survival. The inclusion of probiotics in the FO-0+ diet had no effects on the parameters measured.

Superovulation treatments aim to stimulate multifollicular recruitment, maximizing the number of oocytes or transferable embryos produced. Factors associated with the superovulation protocol, female characteristics and many other factors are determinants in the number and quality of oocytes obtained. An accurate way to assess oocyte quality more precise than morphological appearance is genetic expression. The present study aims to compare the response of nulliparous and multiparous females to superovulatory stimulation, studying its effect on the expression of some genes associated with the activation, growth, development and oocyte-embryo transition of oocytes, as well as its impact on in vivo embryonic development and viability rate at birth. In a first experiment, the effect of stimulation treatment on the ovulation response and the expression of the MSY2, MATER, ITPR1, ITPR2, ITPR3, eIF4E, PAR1, PAPOL-A, PAPOL-G, ZAR1 and YY1 genes in nulliparous and multiparous females were determined. In a second experiment, the implantation and viability at birth of embryos from superovulated nulliparous and multiparous females were analysed. The ovulation rate was significantly higher in the superovulation groups than in the control groups. The ovulation rate was significantly increased in nulliparous females compared with multiparous does. From the eleven genes analysed, only the expression of MATER, PAPOL-A, PAPOL-G and ZAR-1 genes was shown to be different among experimental groups. Finally, in terms of implantation rate and viability at birth, the nulliparous control group showed better results than the rest of the groups. Both hyperstimulation treatment and reproductive female's history seem to alter the transcriptome of important genes related to oocyte maturation and competence acquisition, affecting in vivo embryo viability.

When talking about the techniques of collection, elaboration and construction of adobe and in general of constructions with earth, there is always a great variety of construction techniques, and composition in the blocks that vary according to the region and culture [1], which generates a great uncertainty, and causes that, at present, although we have decree 2113 of 2019, we do not have clarity as to a method and characteristics universally valid and accepted in our country. It is for this reason that in this work we show the modelling of a series of type walls by means of the SAP 2000 program for which applying a static linear analysis for finite elements and an analysis of stresses the service limit states given for a series of walls of different composition are determined, which showed that the percentage of failure for adobe walls is 40% under the given methodology, under this threshold it was determined that the element can be in service and have functionality, exceeding this value it is denoted that the element will lose its functionality without entering into risk of collapse until reaching a percentage of 50%. Finally, the models are obtained with the appropriate effort analyses and design recommendations under the criteria of the results obtained, leaving the possibility of performing an experimental phase to develop the analogy with those raised in the models.

[EN] Preimplantation embryo manipulations during standard assisted reproductive technologies (ART) have significant repercussions on offspring. However, few studies to date have investigated the potential long-term outcomes associated with the vitrification procedure. Here, we performed an experiment to unravel the particular effects related to stress induced by embryo transfer and vitrification techniques on offspring phenotype from the foetal period through to prepuberal age, using a rabbit model. In addition, the focus was extended to the liver function at prepuberal age. We showed that, compared to naturally conceived animals (NC), offspring derived after embryo exposure to the transfer procedure (FT) or cryopreservation-transfer procedure (VT) exhibited variation in growth and body weight from foetal life to prepuberal age. Strikingly, we found a nonlinear relationship between FT and VT stressors, most of which were already present in the FT animals. Furthermore, we displayed evidence of variation in liver function at prepuberal age, most of which occurred in both FT and VT animals. The present major novel finding includes a significant alteration of the steroid biosynthesis profile. In summary, here we provide that embryonic manipulation during the vitrification process is linked with embryo phenotypic adaptation detected from foetal life to prepuberal age and suggests that this phenotypic variation may be associated, to a great extent, with the effect of embryo transfer., This research was funded by Conselleria d'Educacio, Investigacio, Cultura i Esport, Spain, grant number AICO/2019/272. Ximo Garcia-Dominguez was supported by a research grant from the Ministry of Economy, Industry and Competitiveness of Spain (BES-2015-072429).

The construction of civil structures on land has played an important role for centuries, however, due to the seismic requirements and the minimum safety standards that are currently required for any structure, this type of construction has been lagged, it is denoted that the related regulations they are widely dispersed and in most cases. In developed countries, numerous technical and legal problems arise to carry out construction with these materials. In relation to this work, a set of models of raw earth type walls are presented, through the SAP 2000 software, having as a supply of the mechanical properties of this material the Peruvian regulation E.080. For the analysis of these models, a static linear analysis for finite elements and a stress analysis of the service limit state concept were studied. Finally, the models with their respective stress studies, management and design recommendations are presented under the criteria of the analyses carried out, leaving open the possibility of both carrying out an experimental phase to develop the analogy with the postulates and proposed results, as well as such as the option to perform a static pressure analysis by finite elements in order to achieve greater precision and calibration of the model with respect to what can be evidenced in laboratory tests.

The interaction between diet and intestinal health has been widely discussed, although in vivo approaches have reported limitations. The intestine explant culture system developed provides an advantage since it reduces the number of experimental fish and increases the time of incubation compared to similar methods, becoming a valuable tool in the study of the interactions between pathogenic bacteria, rearing conditions, or dietary components and fish gut immune response. The objective of this study was to determine the influence of the total substitution of fish meal by plants on the immune intestinal status of seabream using an ex vivo bacterial challenge. For this aim, two growth stages of fish were assayed (12 g): phase I (90 days), up to 68 g, and phase II (305 days), up to 250 g. Additionally, in phase II, the effects of long term and short term exposure (15 days) to a plant protein (PP) diet were determined. PP diet altered the mucosal immune homeostasis, the younger fish being more sensitive, and the intestine from fish fed short-term plant diets showed a higher immune response than with long-term feeding. Vibrio alginolyticus (V. alginolyticus) triggered the highest immune and inflammatory response, while COX-2 expression was significantly induced by Photobacterium damselae subsp. Piscicida (P. damselae subsp. Piscicida), showing a positive high correlation between the pro-inflammatory genes encoding interleukin 1β (IL1-β), interleukin 6 (IL-6) and cyclooxygenase 2(COX-2)., The research was supported by a grant financed by the Spanish Ministerio de Economía y Competitividad AGL2015-70487-P and Generalitat Valenciana, IDIFEDER/2020/029

[EN] The role of potassium from the seminal plasma and/or the activation media was examined by selectively removing from this media, and by testing the use of channel inhibitors and a K-ionophore. Sperm motility was measured using a CASA system, intracellular K+ and pH were measured by flow cytometry, and sperm head area was measured by ASMA: Automated Sperm Morphometry Analyses. Sperm motility was notably inhibited by the removal of K+ from the seminal plasma and by treatment with the K+ ionophore valinomycin. This therefore indicates that a reduction of K+ levels in the quiescent stage inhibits further motility. The normal decrease in sperm head area induced by seawater activation was altered by the removal of K+ from the seminal plasma, and an increase in the pH; in the quiescent stage was also induced. Intracellular pH (pH;) was quantitatively measured for the first time in European eel spermatozoa, being 7.2 in the quiescent stage and 7.1 post-activation. Intracellular and external pH levels influenced sperm motility both in the quiescent stage and at activation. The alkalinization of the pH; (by NH4Cl) inhibited sperm motility activation, while acidification (by Na-acetate) did not have any effect. Our results indicate that a pH gradient between the sperm cell and the seminal plasma is necessary for sperm motility activation. The presence of the ion K+ in the seminal plasma (or in the extender medium) is necessary in order to maintain sperm volume, intracellular pH and sperm motility., Funded from the SPERMOT project (Spanish Ministry of Science and Innovation, MINECO AGL2010-16009). M.C. Vilchez has a predoctoral grant from UPV PAID Subprogramme 2 (2011-S2-02-6521), Marina Morini has a predoctoral grant from Generalitat Valenciana (Programa Grisolia, GRISOLIA/2012/006), Victor Gallego has a postdoctoral contract from UPV (PAID-10-14), and David S. Penaranda was supported by MICINN (PTA2011-4948-1) and UPV (PTA2011-4948-I). Grants to attend meetings were received from COST Office (AQUAGAMETE COST Action: FA1205).

[EN] The digestive tract, particularly the intestine, represents one of the main sites of interactions with the environment, playing the gut mucosa a crucial role in the digestion and absorption of nutrients, and in the immune defence. Previous researches have proven that the fishmeal replacement by plant sources could have an impact on the intestinal status at both digestive and immune level, compromising relevant productive parameters, such as feed efficiency, growth or survival. In order to evaluate the long-term impact of total fishmeal replacement on intestinal mucosa, the gut mucosa proteome was analysed in fish fed with a fishmeal-based diet, against plant protein-based diets with or without alternative marine sources inclusion. Total fishmeal replacement without marine ingredients inclusion, reported a negative impact in growth and biometric parameters, further an altered gut mucosa proteome. However, the inclusion of a low percentage of marine ingredients in plant protein-based diets was able to maintain the growth, biometrics parameters and gut mucosa proteome with similar values to FM group. A total fishmeal replacement induced a big set of underrepresented proteins in relation to several biological processes such as intracellular transport, assembly of cellular macrocomplex, protein localization and protein catabolism, as well as several molecular functions, mainly related with binding to different molecules and the maintenance of the cytoskeleton structure. The set of downregulated proteins also included molecules which have a crucial role in the maintenance of the normal function of the enterocytes, and therefore, of the epithelium, including permeability, immune and inflammatory response regulation and nutritional absorption. Possibly, the amino acid imbalance presented in VM diet, in a long-term feeding, may be the main reason of these alterations, which can be prevented by the inclusion of 15% of alternative marine sources. Significance: Long-term feeding with plant protein based diets may be considered as a stress factor and lead to a negative impact on digestive and immune system mechanisms at the gut, that can become apparent in a reduced fish performance. The need for fishmeal replacement by alternative ingredients such as plant sources to ensure the sustainability of the aquaculture sector has led the research assessing the intestinal status of fish to be of increasing importance. This scientific work provides further knowledge about the proteins and biologic processes altered in the gut in response to plant protein based diets, suggesting the loss of part of gut mucosa functionality. Nevertheless, the inclusion of alternative marine ingredients was able to reverse these negative effects, showing as a feasible option to develop sustainable aquafeeds., The first author was supported by a contract-grant (Contrato Pre doctoral para la Formacion de Profesorado Universitario) from Subprogramas de Formacion y Movilidad within the Programa Estatal de Promocion del Talento y su Empleabilidad of the Ministerio de Educacion, Cultura y Deporte of Spain.

[Background]: In order to ensure sustainability of aquaculture production of carnivourous fish species such as the gilthead seabream (Sparus aurata, L.), the impact of the inclusion of alternative protein sources to fishmeal, including plants, has been assessed. With the aim of evaluating long-term effects of vegetable diets on growth and intestinal status of the on-growing gilthead seabream (initial weight = 129 g), three experimental diets were tested: a strict plant protein-based diet (VM), a fishmeal based diet (FM) and a plant protein-based diet with 15% of marine ingredients (squid and krill meal) alternative to fishmeal (VM+). Intestines were sampled after 154 days. Besides studying growth parameters and survival, the gene expression related to inflammatory response, immune system, epithelia integrity and digestive process was analysed in the foregut and hindgut sections, as well as different histological parameters in the foregut., [Results]: There were no differences in growth performance (p = 0.2703) and feed utilization (p = 0.1536), although a greater fish mortality was recorded in the VM group (p = 0.0141). In addition, this group reported a lower expression in genes related to pro-inflammatory response, as Interleukine-1β (il1β, p = 0.0415), Interleukine-6 (il6, p = 0.0347) and cyclooxigenase-2 (cox2, p = 0.0014), immune-related genes as immunoglobulin M (igm, p = 0.0002) or bacterial defence genes as alkaline phosphatase (alp, p = 0.0069). In contrast, the VM+ group yielded similar survival rate to FM (p = 0.0141) and the gene expression patterns indicated a greater induction of the inflammatory and immune markers (il1β, cox2 and igm). However, major histological changes in gut were not detected., [Conclusions]: Using plants as the unique source of protein on a long term basis, replacing fishmeal in aqua feeds for gilthead seabream, may have been the reason of a decrease in the level of different pro-inflammatory mediators (il1 β, il6 and cox2) and immune-related molecules (igm and alp), which reflects a possible lack of local immune response at the intestinal mucosa, explaining the higher mortality observed. Krill and squid meal inclusion in vegetable diets, even at low concentrations, provided an improvement in nutrition and survival parameters compared to strictly plant protein based diets as VM, maybe explained by the maintenance of an effective immune response throughout the assay., The research has been partially funded by Vicerrectorat d’Investigació, Innovació i Transferència of the Universitat Politècnica de València, which belongs to the project Aquaculture feed without fishmeal (SP20120603)

Since the first human was conceived through in vitro fertilisation in 1978, over 6.5 million babies have been born by assisted reproductive technologies (ARTs). Although most ART babies and children seem healthy, in recent years several studies have evidenced a potential impact of ARTs on long-term development and health. Herein, we have developed an animal model to determine whether vitrified embryo transfer procedure induces phenotypic changes over the growth performance and in the complementary transcriptomic and proteomic analyses at hepatic level. To this end, 2 populations were developed; vitrified embryos transferred to the surrogate mothers (VT) and naturally conceived animals (NC). After delivery, animals were weighed weekly from 1 to 20 weeks of age. In adulthood, animals were euthanized and organs were harvested and weighed. After that, liver tissue was used to identify changes in the transcriptomic and proteomic profile. At adulthood, VT group showed significant lower body, liver and heart weight. After functional analysis of RNA-Seq data, a subset of 96 differentially expressed transcripts in VT animal were related to alteration in zinc homeostasis, lipid metabolism, and hepatic immune pathways. After proteomic analysis, a subset of 76 differentially expressed proteins also revealed some disturbed metabolic pathways related with the lipid and glycan metabolism, and an impaired oxidative metabolism related to ATP synthesis in the mitochondria. Current findings suggest that progeny derived after transfer of vitrified embryos have long-term consequences on growth rate and vital organs weights in adulthood, correlated with molecular signatures at transcriptomic and proteomic level of hepatic tissue.

[EN] Academic procrastination is a fact related to the delay or postpone of academic work until last minute. This phenomenon is evident in a vast majority of university students, and its occurrence is increasing. In order to analyse possible causes and/or solutions, we studied if longer time for accomplishing an assignment incentives or avoids procrastination among university students. Results showed that both short and long time-frame groups tended to procrastinate in the same way. Additionally, academic grades did not revealed differences between groups, as the procrastination was the same between groups. Thus, this study shows that even with longer period of time to accomplish a task, university students tend to procrastinate, and thus seem to have a negative effect on their assignment grades. Therefore, it seems a current problem and measures should be developed in order to solve it, [ES] La procrastinación académica es un término que hace referencia a la postergación o posposición de cualquier actividad o tarea académica para el último minuto. Este fenómeno prevalece de forma mayoritaria entre los estudiantes universitarios, y su incidencia parece ir en aumento. Con el fin de analizar las posibles causas y/soluciones a la procrastinación, en este trabajo se estudió si un mayor tiempo para la elaboración y/o desarrollo de las actividades y tareas universitarias impedía o incentivaba la procrastinación entre los estudiantes universitarios. Los resultados mostraron que tanto los grupos con menor tiempo para el desarrollo de tareas, como los grupos con un mayor tiempo tendían a procrastinar de la misma manera. Además, las notas medias de las tareas no mostraron diferencias entre los grupos dado que la procrastinación fue la misma en ambos grupos. Por ello, este estudio muestra que incluso con periodos más prolongados para la elaboración de la actividad, los estudiantes universitarios tienden a procrastinar, y este hecho parece tener un efecto negativo sobre la nota de la actividad. En conclusión, la procrastinación es un grave problema en el sistema universitario actual y deberían tomarse medidas para solucionarlo., Este proyecto ha sido financiado por el Vicerrectorado de Estudios, Calidad y Acreditación de la Universidad Politécnica de Valencia (UPV) en el marco del Programa Proyectos de Innovación y Mejora Educativa (PIME/2017/B/010) y la Escuela de Ingeniería Agrícola y Medio Ambiente ( ETSIAMN) de la Universitat Politècnica de València (UPV).

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka´s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

[EN] The present study was undertaken to evaluate the correlations between the fatty acids in the liver and testis and the plasma levels of the hormonal steroids used during eel spermatogenesis, in order to clarify the physiological roles fatty acids play in the spermatogenetic process. The stages of testis development (S1-S5) were assessed by histological observations in order to classify the different phases of hormonally-induced spermatogenesis and evaluate the possible relationships between the hormones and fatty acids in each stage. The highest plasma levels of 17 beta-estradiol (E2), testosterone (T) and 11-ketotestosterone (11KT) were found in S1, when spermatogonial proliferation occurs. A correlation was found between 17 alpha-20 beta-dihydroxy-4-pregnen-3- one (DHP) levels and some fatty acids during the proliferation and growing phases (S1-2), suggesting that DHP might modulate lipid metabolism in the liver during early spermatogenesis. The DHP levels increased significantly during the growing phase (S2) and remained at high levels throughout the subsequent development stages (S3-S5). Similar to results found in mammals, our results show that in the eel there are regulatory mechanisms, including eicosapentaenoic acid (20:5-n3, EPA) and docosahexaenoic acid (22:6-n3, DHA), which act as modulators in the synthesis of androgens, particularly during the final phase of sperm maturation. Our results suggest that the fact that EPA, ARA and DHA concentrations in the eel testis remain constant/stable during spermiation could be related to the subsequent union of the spermatozoa and the egg. The findings from this research provide new insights for further studies about the possible effect of steroids on desaturase activity and highlight the importance of the effect of lipid metabolism during male eel spermatogenesis. (C) 2014 Elsevier B.V. All rights reserved., Funded by the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no. 245257 (PRO-EEL). D. S. P. had a contract co-financed by MICINN and UPV (PTA2011-4948-I) and received a Shortterm Scientific Mission grant from COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE) to carry out the steroids analyses in Norway.

article i nfo Wild female European eels were matured with CPE (carp pituitary extract) under three thermal regimes, two of which were variable (T10-15 and T15-18, moving from 10 to 15 °C and from 15 to 18 °C, respectively) and one constant, at 18 °C (T18). Before and during hormonal treatment, the eels were sampled and biometric measure- ments were taken. Immunoassays of sex steroids and vitellogenin were performed, as well as qPCR analyses of gene expression (ovarian cyp19a1) and ovarian histology. Prior to the hormonal treatment, the silver eels which had been maintained at 18 °C showed lower 11-KT and E2 plasma levels compared to those maintained at 10 °C. In addition, in the early vitellogenic stage, the androgen and cyp19a1 levels were lower at 18 °C than at 10 °C. Both these results and the positive correlations found between GSI and 11-KT (at the PV stage) and between oocyte diameter and cyp19a1 levels (in the EV stage) suggest that early ovarian development is facili- tated at low temperatures. Vitellogenesis was induced by CPE in all the thermal groups, but progression to the mid-vitellogenic stage was only observed after an accumulation of 900-1200 °D, at 15 or 18 °C, and progression to the late vitellogenic stage was only observed after an accumulation higher than 1300 °D, at 18 °C. Although temperature increased the rate of CPE-induced ovarian development, our results clearly indicate that this in- crease is not linear, but exponential, with acceleration in the increase of GSI at 18 °C from the mid-vitellogenic stage, or after an accumulation of 1300 °D. For the first time, a down-regulation of ovarian cyp19a1 caused by high temperatures in CPE-treated eels was observed. These results demonstrate that temperature can modulate eel ovarian development both before and after exogenous hormonal stimulations, and this knowledge could be used to manipulate the timing of vitellogenesis progression under laboratory conditions.

[EN] Paralogues pairs are more frequently observed in eels (Anguilla sp.) than in other teleosts. The paralogues often show low phylogenetic distances; however, they have been assigned to the third round of whole genome duplication (WGD), shared by all teleosts (3R), due to their conserved synteny. The apparent contradiction of low phylogenetic difference and 3R conserved synteny led us to study the duplicated gene complement of the freshwater eels. With this aim, we assembled de novo transcriptomes of two highly relevant freshwater eel species: The European (Anguilla anguilla) and the Japanese eel (Anguilla japonica). The duplicated gene complement was analysed in these transcriptomes, and in the genomes and transcriptomes of other Actinopterygii species. The study included an assessment of neutral genetic divergence (4dTv), synteny, and the phylogenetic origins and relationships of the duplicated gene complements. The analyses indicated a high accumulation of duplications (1217 paralogue pairs) among freshwater eel genes, which may have originated in a WGD event after the Elopomorpha lineage diverged from the remaining teleosts, and thus not at the 3R. However, very similar results were observed in the basal Osteoglossomorpha and Clupeocephala branches, indicating that the specific genomic regions of these paralogues may still have been under tetrasomic inheritance at the split of the teleost lineages. Therefore, two potential hypotheses may explain the results: i) The freshwater eel lineage experienced an additional WGD to 3R, and ii) Some duplicated genomic regions experienced lineage specific rediploidization after 3R in the ancestor to freshwater eels. The supporting/opposing evidence for both hypotheses is discussed., This study received funding from the project REPRO-TEMP (AGL2013-41646-R) funded by the Spanish Ministry of Economy and Competitiveness, and from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS), which also included the predoctoral contracts of CR and JGHJ. VG has a postdoctoral grant from the Spanish Ministry of Science, Innovation and Universities (MICIU; Programa Juan de la Cierva-Incorporacion; IJCI-2017-34200). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Several studies have extensively examined structural and biochemical damage induced by cryopreservation that may lead to loss of rabbit embryo viability, but very little information is available on alterations in growth during gestation and at gene expression level. We started our work by comparing the distribution of losses of embryo and foetal development between control and vitrified rabbit morulae. Furthermore, data on foetal sack, foetal and maternal placenta and foetus size for 10-14 days of gestation were evaluated by ultrasonography. We reported that vitrification procedure causes detrimental effects on rabbit embryo and foetal development, with two major peaks of losses: one before the implantation (at day 6) and the other during the second part of gestation (after day 14). However, foetal loss may occur during the implantation process and placenta development, as there was a reduction in development of foetus produced from vitrified-warmed embryos between day 10 and 14 of gestation. For these reasons, using a recent microarray study performed in frozen-thawed rabbit embryos as a point of reference, we analysed the effects of vitrification procedure on the expression of 10 candidate genes in 6-day-old blastocysts obtained after vitrification and transfer. We observed that the relative expressions of mRNA transcripts from SCGB1A1, EMP1, ANXA3 and EGFLAM genes were significantly altered. This could help explain why a large number (29%) of vitrified embryos were successfully implanted but subsequently failed to develop to term. Further studies in subsequent embryo-foetal developmental stages, such as initiation of placenta formation, together with more sensitive high-throughput tools, should help us understand the deficiencies that hinder foetal development and identify the repairing mechanism employed by embryos to overcome vitrification effects. (C) 2013 Elsevier Inc. All rights reserved.

[EN] Young rabbit females selected for growth rate may have nutritional needs, which may not be met with the common practice of feed restriction during rearing in commercial rabbit production. The aim of this study was to analyse whether two different feeding programmes: ad libitum or restricted (130 g/day) feeding, applied in young rabbit females for 1 month at the end of rearing, could modulate the origin of ovulation process and the quality of the oocytes. At 16 weeks of age, 34 females were randomly assigned to restricted or ad libitum feeding, maintaining these conditions for a month. Then, in an initial experiment, transcriptional profiling of hypothalamus-hypophysis tissue was performed to assess failure to ovulate. In the second experiment, the gene expression analysis of some candidate genes related to oocytes quality was performed. Our results demonstrated that neither of the two feeding programmes modified the transcription of hypothalamus-hypophysis tissue, while the only differences in MSYR expression were found in in vivo mature oocytes ready for successful fertilization. Specifically, MSYR was over-expressed in oocytes from females fed ad libitum. MSYR is one of the most abundant proteins in the oocyte and has proven to be a key regulator of maternal RNA transcription and translation. This finding suggests that MSYR gene is a promising gene in our understanding of the relationship between high growth rate and reproductive performance decline., This work was supported by the Spanish Research Projects AGL2014-53405-C2-P and AGL2011-30170-C02-01 (CICYT). Carmen Naturil was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2013/296).

[EN] Estradiol (E-2) can bind to nuclear estrogen receptors (ESR) or membrane estrogen receptors (GPER). While mammals possess two nuclear ESRs and one membrane GPER, the European eel, like most other teleosts, has three nuclear ESRs and two membrane GPERs, as the result of a teleost specific genome duplication. In the current study, the expression of the three nuclear ESRs (ESR1, ESR2a and ESR2b) and the two membrane GPERs (GPERa and GPERb) in the brain-pituitary-gonad (BPG) axis of the European eel was measured, throughout spermatogenesis. The eels were first transferred from freshwater (FW) to seawater (SW), inducing parallel increases in E2 plasma levels and the expression of ESRs. This indicates that salinity has a stimulatory effect on the E-2 signalling pathway along the BPG axis. Stimulation of sexual maturation by weekly injections of human chorionic gonadotropin (hCG) induced a progressive decrease in E-2 plasma levels, and different patterns of expression of ESRs and GPERs in the BPG axis. The expression of nuclear ESRs increased in some parts of the brain, suggesting a possible upregulation due to a local production of E-2. In the testis, the highest expression levels of the nuclear ESRs were observed at the beginning of spermatogenesis, possibly mediating the role of E2 as spermatogonia renewal factor, followed by a sharply decrease in the expression of ESRs. Conversely, there was a marked increase observed in the expression of both membrane GPERs throughout spermatogenesis, suggesting they play a major role in the final stages of spermatogenesis., Funded by the Spanish Ministry of Science and Innovation (REPRO-TEMP project; AGL2013-41646-R) and IMPRESS (Marie Sklodowska-Curie Actions; Grant agreement no: 642893). M.C. Vilchez has a predoctoral grant from UPV PAID Programme (2011-S2-02-6521), M. Morini has a predoctoral grant from Generalitat Valenciana (Programa Grisolia). D.S. Penaranda was supported by MICINN and UPV (PTA2011-4948-1).

[EN] Vertebrate eggs are surrounded by an extracellular glycoprotein coat termed zona pellucida (ZP). Integrity of ZP is critical for a correct embryo development. Two zona pellucida protein genes (zpb and zpc) from European eel were characterized, specific qPCR assays developed and their expression in immature males and females carried out. An experimental group of silver-stage eel females was maintained at 18 C and hormonally induced to sexual maturation by weekly injections of carp pituitary extract during 12 weeks. Changes in zpb and zpc expression during sexual maturation were studied in liver and ovary by qPCR. In liver, no changes were recorded during hormonal treatment, while in ovary expression of both genes decreased during sexual development. These results are a first step in the characterization of ZP in European eel and in the understanding of the mechanism underlying egg envelope formation., Funded from the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", Grant agreement no. 245257 (Pro-Eel) and Generalitat Valenciana through the GV/2007/202 project and ACOMP/2011/229. D.S. Penaranda has a postdoc Grant from the Universitat Politecnica de Valencia (CE-01-10). I.M. and V.G. have predoctoral Grants from Generalitat Valenciana and Spanish MICINN, respectively. F.-A. Weltzien received funding from The Norwegian School of Veterinary Science.

[EN] Since 1960, the European eel (Anguilla anguilla) has suffered a dramatic reduction in natural stocks. Breeding in captivity is considered an alternative, but obtaining high quality sperm seems basic on this regard. The main objective of this study was to assess the effects of three thermal regimes (two of them variable: T10 and T15; and one of them constant: T20) and three hormonal treatments with different hormones (hCG, hCGrec and PSMG) on the induction of maturation in European eel males. In the case of the thermal regimes, our results demonstrated that the onset and progression of spermiation are strongly influenced, and perhaps closely regulated, by water temperature. T20 demonstrated the best results in all the sperm parameters (volume, density, motility, kinetic features, etc.) throughout most weeks of treatment, becoming a reliable and productive method for inducing spermiation in this species. In the case of hormonal treatments, the onset and progression of spermiation in European eel males were influenced by the hormone used. In this respect, hCGrec produced the best results in all the sperm parameters including volume, density, motility, kinetic features, etc., throughout most weeks of treatment, thus becoming an effective alternative treatment to the standard hCG treatment used to induce spermiation in eel species. Moreover, hCGrec gave rise to the best economical profitability, making it possible to obtain good quality sperm samples at a lower price than by using the other two hormonal treatments. © 2012 Elsevier B.V. All rights reserved Guardar / Salir Siguiente >, Funded by the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no. 245257 (PRO-EEL) and Generalitat Valenciana (ACOMP/2012/086). Victor Gallego, Ilaria Mazzeo and M. Carmen Vilchez have predoctoral grants from the Spanish Ministry of Science and Innovation (MICINN), Generalitat Valenciana, and UPV PAID Programme (2011-S2-02-6521), respectively. Paulo C.F. Carneiro had post-doc grants from PAC-EMBRAPA and UPV PAID Programme (PAID-02-11). David S. Penaranda was supported by a contract co-financed by MICINN and UPV (PTA2011-4948-I).

Parthenote embryos are being considered as an alternative source of embryonic stem cells. However, as there is still a dearth of knowledge of this kind of embryos, a better understanding of their biology is needed for their application. In this work, we studied the differences and similarities between parthenotes and normal embryos at the blastocyst stage in vivo developed. We analysed the expression of factor OCT-4, vascular endothelial growth factor (VEGF), insulin-like growth factor I (IGF-I) and uteroglobin (UG) by real-time PCR. To do so, oocytes were recovered and after activation procedure were transferred by ventral middle laparoscopy to receptive does to undergo completely in vivo development. Does were slaughtered 6 days post-ovulation induction, and parthenote and normal embryos were recovered for mRNA expression analysis. Our results reported that parthenotes and normal embryos showed similar mRNA expression for OCT-4 and VEGF. However, IGF-I and UG showed to be over-expressed in parthenote embryos. Thus, our study highlights that despite the in vivo development of parthenotes, they still seem to have an altered expression and, therefore, to be different to normal embryos. The altered expression pattern of parthenote embryos suggests that these embryos should be studied carefully before future application.

[EN] The development of powerful software has made possible spermatozoa morphology studies. However, some problems have emerged in relation to protocol standardization to compare results from different laboratories. This study was carried out to compare two techniques commonly used (staining vs phase contrast technique) for the morphometry study of gilthead sea bream spermatozoa using an integrated sperm analysis system (ISAS). Spermatozoa morphometry values were significantly affected by the technique used, and phase contrast technique was found to be the more accurate method, showing lower coefficients of variation on spermatozoa morphometry parameters measurements. Moreover, it has been shown that cryopreservation process produces damage in gilthead sea bream spermatozoa, causing negative effects in sperm parameters as spermatozoa morphometry (a decrease in cell volume), motility (from 95 to 68% motile cells) and viability (from 95 to 87% of live cells), being the addition of freezing medium containing cryoprotectant (DMSO) an important factor that caused the morphometry changes. (C) 2012 Elsevier Inc. All rights reserved., This work was financed by the Spanish Ministry of Science and Innovation (MICINN; AGL2007-64040-C03-00, Project SELECTBREAM). V. Gallego and I. Mazzeo were supported by predoctoral scholarships financed by the Spanish MICINN and Generalitat Valenciana, respectively. D.S. Penaranda had a postdoctoral grant from UPV.

To obtain more reliable results with biological significance, it requires data normalization using an appropriate internal control gene. In rabbits, there are classic stable reference genes that have been identified for normalization in oocytes and pre-implantation stage embryos. However, effects of embryonic genotype on reference gene selection have not been elucidated. The aim of this study was to test (i) the stability of mRNA transcription level for histone (H2afz) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes in rabbit blastocysts from two lines selected by different criteria (litter size and post-weaning daily weight gain) and (ii) its influence on biological significance examined by means of a set of embryonic transcripts, such as POU5F1 (Oct-4), epidermal growth factor receptor (erbB3), transforming growth factor-beta2, vascular endothelial growth factor and gamma interferon (Ifn-gamma). The geNorm, NormFinder and BestKeeper programs showed similar results, pointing out that H2afz and GAPDH were the most stable reference genes in rabbits selected on litter size at weaning. Moreover, our study revealed that embryonic genotype affected target gene expression when a single reference gene was used to analyse mRNA expression in blastocysts. Results showed that GAPDH gene is better than H2afz for gene expression studies of both embryo genotypes. A normalization factor derived from H2afz and GAPDH is likely to be appropriate when RT-qPCR was performed in rabbit embryos with different genotypes.

Gestation is a complex process that involves different growth factors, cytokines and adhesion proteins related with embryo development, cellular differentiation and proliferation, embryo-endometrium interaction, angiogenesis, maternal-embryonic recognition and growth development of placenta and embryos. In this study, we examine pre-implantational (at 6 days of gestation) and gestational (at 12 days and total from ovulation to birth) losses in two rabbit lines selected by different criteria (post-weaning daily gain and litter size) and the pattern of a set of candidate transcripts, at 6 days of gestation, related with embryo development and implantation process, such as Oct-4, epidermal growth factor receptor 3 (erbB3), Transforming Growth Factor β2, Vascular Endothelial Growth Factor and Interferon γ and related with insulin-like growth factors signalling as insulin growth factors I and II and their receptors in rabbit blastocysts and endometrial tissue. Similar pre-implantational losses were obtained in both lines. However, the gestational losses of the line selected by post-weaning daily gain clearly mirrored an increase in losses by 50% at 12 days and at birth (22.4 vs 9.5 and 50.2 vs 25.4, respectively, between line selected by post-weaning daily gain and line selected by litter size). In blastocysts and endometrial tissue at 6 days of gestation qRT-PCR assays indicated that the mean insulin-like growth factor (IGF)-IIR mRNA expression was down-regulated in line selected by post-weaning daily gain. Dysregulation of the IGF-IIR could be potential reasons for induced gestational losses. We conclude that IGF-IIR gene expression in blastocyst and endometrial tissue at 6th day of gestation tends to decline in line selected by post-weaning daily gain. The functional significance related with gestational losses is uncertain.

To study genes involved in embryo developmental competence and implantation in rabbits, the expression of a panel of genes related to pluripotency, angiogenesis, proliferation, apoptosis and differentiation were evaluated in late rabbit blastocysts. Thirty nulliparous does were used to obtain a total of 184 in vivo-derived blastocysts on days 4, 5 and 6 of development. The relative transcript abundance of vascular endothelial growth factor (VEGF), epidermal growth factor receptor 3 (erbB3), transforming growth factor β2 (TGF β2) and transcription factor OCT-4 were analysed from eight pools of each stage of development, using quantitative real-time reverse transcriptase-polymerase chain reaction (qrtRT-PCR). mRNA expression was detected for all genes in 4-, 5- and 6-day-old blastocysts, according to blastocyst growth and implantation proximity. Significant differences in OCT-4, VEGF and TGF β2 expression were observed between days of development. Results show a down-regulation of OCT-4 from the 4th day, contrasting with the up-regulation of VEGF and TGF β2 at 6-day-old blastocyst. These findings corroborate the importance of VEGF and TGF β2 in rabbit embryo development and implantation and suggest a possible regulator role of OCT-4 in embryonic angiogenetic factors. On the other hand, no differences were found for erbB3 expression. Therefore, the study of specific gene transcripts in rabbit blastocyst could provide novel embryo developmental competence markers and might be used as a new tool for further studies of embryo quality and in vitro development.

Summary In order to study different storage conditions, dilution ratios and additives for fresh European eel sperm were assayed to determinate the optimum conditions for short-term storage. Two experiments were conducted, analysing the sperm motility and its viability at different times of incubation (1, 4, 7, 11 and 14 days). In the first part of the work, 19 pooled samples were used, and seven in the second experiment. Firstly, exposure to atmospheric and air-limited conditions were compared, registering the highest motility under air-limited conditions, showing no differences to the performance of fresh sperm samples until 96 h of incubation. Also, two different dilutions were checked, with ratios 1 : 10 and 1 : 50, with the latter obtaining better results. A second experiment was performed trying to improve the results obtained in the first experiment. Using air-limited condition and 1 : 50 dilution ratio, the effect of different additives (foetal bovine serum, FBS; N-Tris(hydroxymethyl) methyl-3-aminopropanesulfonic Acid, TAPS; bovine serum albumin, BSA; Glucose) were evaluated. The sperm diluted in the media containing glucose provided motilities without difference in comparison to fresh samples during first week of incubation. At 7 days of storage, the medium containing 300 mm glucose was the only one causing higher motility (77.9 ± 2.5%) in comparison with control medium (50.1 ± 11.5%), demonstrating that the sugar addition was positive to preserve the sperm movement. In conclusion, a new method of eel sperm preservation has been developed, using air-limited conditions, 1 : 50 dilution ratio and glucose as an additive, which provided motilities without significant differences until 7 days of incubation.

Summary Along with sperm quality parameters, the protein profile of European eel seminal plasma was analyzed during induced spermiation (n = 56 samples). Motility, Percentage of live cells, spermatozoa head morphometry and concentration showed low values during the initial weeks of spermiation and maintained high levels throughout the rest of the experiment. The protein profile gradient by SDS-PAGE (4–15%) registered four important electrophoretic bands around 80, 40, 26 and 12 KDa. Three of them showed significant differences in concentration during treatment (80, 40 and 12 KDa), and all of them showed the highest value on the 8th week. Both 80 and 12 KDa bands increased until the 8th week, followed by a progressive decline. One possible explanation for these profiles is that, in the first weeks of treatment, proteins originated from blood plasma are accumulated in the seminal plasma, and from the 8th week some of these proteins are incorporated into the spermatic membranes. The 40 KDa protein band also increased during the first 8 weeks, but maintained high concentrations in the seminal plasma for the rest of the experiment. One result confirms the theory that the presence of proteins in the seminal plasma having a molecular weight lower than 50 KDa increased spermatozoa motility, since the 40 KDa band displayed significantly higher values coinciding with the high percentages of spermatozoa motility. Seminal plasma proteins seem to have an important role in spermatogenesis and spermatozoa movement, but further studies are necessary to discover the identity of these proteins and their precise functions.

Summary As no studies have been carried out to investigate the role of fatty acids in male European eel (Anguilla anguilla) maturation, the main aim of this work was to determine which fatty acids could play a central role in sexual maturation. To this end, European eel males were hormonally induced to sexual maturation and variations in fatty acid composition in muscle, liver, testis and milt were studied. Eels were injected weekly with hCG (Human Chorionic Gonadotropin) for 13 weeks and 10 eels per week were sacrificed. Histological analysis was performed to determine the spermatogenic stage of each male. In muscle, no variations or preferential utilization of fatty acids were detected. In liver, MUFAs (Monounsaturated Fatty Acids) were the most abundant fatty acids in the first weeks, while in the last week PUFAs (Polyunsaturated Fatty Acids) showed the highest concentrations. In testes, PUFAs content increased due to DHA (Docosahexaenoic acid), EPA (Eicosapentaenoic acid) and AA (Arachidonic acid) accumulation, while MUFAs diminished especially due to oleic and palmitoleic acid decrease. Linolenic (EPA and DHA precursor) and linoleic (AA precursor) acids also decreased. As evidenced by histology, main changes in testes matched the appearance of spermatids and first spermatozoa, at the end of meiotic divisions. In milt, no variations were appreciated and PUFAs were the most abundant fatty acids, close to 50%. Due to their increase in testes and the high concentration in milt, PUFAs probably play an important role in the sexual maturation of European eel. Further research is necessary to determine the relationship between feeding, fatty acids and sperm quality.

Contents The sperm of European eel shows a high density and the time of spermatozoa motility is very short after activation with sea water. These characteristics make difficult the sperm handling and its quality assessment. Several diluents were previously described for the Japanese eel obtaining over 3 weeks’ conservation times under refrigeration, but they rendered bad results in the European species. In the present study, several diluents were developed taking as basis the P1 medium, and using different dilution ratios (1 : 50, 1 : 100) and two pH (6.5, 8.5). The effect of the addition of bovine serum albumin (BSA, 2% w/v) was also evaluated. At 24 h, undiluted samples already showed significant lower motility and viability than sperm samples diluted in the different media. The results for diluents with pH 6.5 and 8.5 were different. Spermatozoa diluted in media at pH 6.5 cannot be activated at 24 h, while samples diluted in the diluents with pH 8.5 and added with BSA did not show significant differences with respect to the fresh sperm motility until 48 h. The viability (percentage of alive cells) did not show differences until 1 week, independent of the dilution ratio. After 1 week, the motility was approximately 30% in the media containing BSA, which presented no differences for head size of the spermatozoa (perimeter and area) until 72 h and 1 week, respectively. In conclusion, the combination of one medium having similar physico-chemical characteristics to the seminal plasma, including pH 8.5, and supplemented with BSA can be used in different dilution ratios for the sperm’s short-term storage, preserving its motility capacity.

Summary The aim of this study was to evaluate the effects of different extenders on sea bass (Dicentrarchus labrax) spermatozoa motility and morphology. Six sperm extenders based on inactivator media, DI1 (here named SAN) and Non-Activating Medium (NAM) were tested with European sea bass spermatozoa. The best results were obtained with NAM medium (59.83 mm NaCl, 12.91 mm MgCl2, 1.47 mm KCl, 3.51 mm CaCl2, 20 mm NaHCO3, 0.44 mm glucose) plus 1 and 2% of BSA (NAM1 and NAM2, respectively). The motility of the spermatozoa incubated in those media was similar to the fresh sperm until 48 h (NAM1: 74.3 ± 5.4; NAM2: 78.8 ± 5.8%, and higher than undiluted sperm, 19.1 ± 7.8). We also checked the spermatozoa motility and morphology reactions with some of the best extenders, NAM2 and SAN, and combined them with different concentrations (2, 5, 10%) of three cryoprotectants: methanol, glycerol and dimethyl sulphoxide (DMSO). Glycerol + SAN or NAM2 caused activation of spermatozoa motility, which was lost 5 min later. Methanol and DMSO plus NAM2 extenders resulted in a low activation level and high motility 5 min after incubation, identifying these combinations as good candidates to be used in the cryopreservation of the European sea bass spermatozoa.

[EN] This study evaluates the effects of temperature on hCG-induced spermatogenesis in European eel (Anguilla anguilla), subjected to three thermal regimes: T10: 10 degrees C (first 4 weeks), 15 degrees C (next 3 weeks) and 20 degrees C (last 6 weeks); T15: 15 degrees C (first 4 weeks) and 20 degrees C (last 9 weeks); and T20: constant 20 degrees C for the duration of the experiment. At 10 degrees C, maturation stopped in the A spermatogonial stage (SPG1), and no further maturation was observed until the temperature was >= 15 degrees C. With the aim of explaining these results, the influence of temperature on steroidogenic enzyme gene expression and steroid synthesis was tested. The initial synthesis of androgens (T and 11-KT) increased at SPG1, and was not influenced by temperature. Likewise, the gene expression of the steroidogenic enzymes linked to androgen synthesis (aacyp11a1, aacyp17-I and aa11 beta HSD) also increased at SPG1. In contrast, no correlation was seen between the increase in E2 and the aacyp19a1 gene expression peak in the testes, with E2 increasing as a consequence of the seawater acclimation carried out before hormonal treatment, and peaking the aacyp19a1 gene expression at B spermatogonial stage (SPG2).Aacyp21 gene expression was also higher at SPG2, and this stage was only reached when the rearing temperature was >= 15 degrees C. In conclusion, androgen synthesis is not dependent on temperature, but further maturation requires higher temperatures in order to induce a change in the steroidogenic pathway towards estrogen and progestin synthesis. This study demonstrates that temperature plays a crucial role in European eel maturation, even perhaps controlling gonad development during the reproductive migration. (C) 2016 Elsevier Inc. All rights reserved., This study was funded by the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no245257 (PRO-EEL), VLC/CAMPUS Program (SP.20140630) and by the MINECO (REPRO-TEMP; AGL2013-41646-R). V.G. and I.M. had predoctoral grants from MINECO (BES-2009-020310) and Generalitat Valenciana, respectively. M.C.V. and M.M. have predoctoral grants from UPV (2011-S2-02-6521) and Generalitat Valenciana (Programa Grisolia), respectively. D.S.P. was supported by MICINN and UPV (PTA2011-4948-I) and was granted with a Short-Term Scientific Mission to make the steroids analyses in Tromso by COST Office (COST Action FA1205: Assessing and improving the quality of aquatic animal gametes to enhance aquatic resources. The need to harmonize and standardize evolving methodologies, and improve transfer from academia to industry; AQUAGAMETE).

Activation at fertilization of the vertebrate egg is triggered by Ca 2 + waves. Recent studies suggest the phospholipase C zeta (PLCζ), a sperm-specific protein, triggers egg activation by an IP3-mediated Ca 2 + release and allow Ca 2 + waves at fertilization. In the present study we cloned, characterized, and phylogenetically positioned the European eel PLCζ (PLCζ1). It is 1521bp long, with 10 exons encoding an open reading frame of 506 amino acids. The amino acid sequence contains an EF-hand domain, X and Y catalytic domains, and a carboxy-terminal C2 domain, all typical of other PLCζ orthologous. The tissue distribution was studied, and the gene expression was determined in testis during induced sexual maturation at three different thermal regimes. Also, brain and pituitary expression was studied through sex maturation at constant temperature. plcζ1 was expressed in brain of male and female, in testis but not in ovaries. By first time in vertebrates, it is reported plcζ1 expression in the pituitary gland. Testis plcζ1 expression increased through spermatogenesis under all the thermal regimes, but being significantly elevated at lower temperatures. It was very low when testis contained only spermatogonia or spermatocytes, while maximum expression was found during spermiogenesis. These results support the hypothesis for an eel sperm-specific PLCζ1 inducing egg activation, similarly to mammals and some teleosts, but different from some other teleost species, which express this protein in ovaries, but not in testes.

Fish sperm quality has traditionally been estimated by subjective evaluation of motility and sperm concentration. Alternative methods for evaluation of sperm quality have been developed in the last decade and enable estimation of spermatozoa head morphometry, membrane integrity and mitochondrial function. Weekly injections of human chorionic gonadotropin (hCG) induced spermiation in farmed male European eels. The milt volume increased from the 5th to 12th weeks. Sperm concentration significantly increased from the 5th week, reaching the highest values at the 8th week, while best motility results were registered at the 9th week of treatment. Coinciding with these intervals, the percentage of dead spermatozoa determined with Hoechst staining showed a reduction in the 8th to 11th weeks of treatment, while the percentage of mitochondrial functionality determined by JC-1 staining did not show a similar pattern. The automatic sperm morphology analysis (ASMA) of the spermatozoa head length, width, area and perimeter showed a significant growth from the 5th to 8th weeks. However, the analysis of isolated descriptive parameters may be difficult to understand because there is a variability in these parameters for each week, making knowledge of the growth kinetic complex. The global size of the spermatozoa head was calculated by applying principal component analysis (PCA), because this method establishes new components that make the interpretation of results easier, allowing a whole interpretation of the changes in the cell morphology. PC1 defines the global head size and shows a significant increase between the 5th and 8th weeks of treatment, showing shorter changes until 12th week. PC2 shows a significant increase in the spermatozoa width from the 5th to 7th weeks. Considering the results of the variations in the principal components defining European eel spermatozoa morphometry, it may be concluded that hCG maturative treatment produced thick cells during the first weeks of spermiation, and subsequent samplings showed an increase in cell width and length. These changes in sperm morphometry coincide with the highest sperm quality assessed as sperm motility and concentration, as well as with the best results obtained in previous studies reporting the best sperm quality between weeks 8 and 10 of hCG treatment. These results support the use of ASMA and Hoechst staining techniques as alternative methods for the evaluation of fish sperm quality.

Five hormonal treatments with human chorionic gonadotropin (hCG) were tested for the induction of maturation and spermiation in male farmed eels. The main aim was to optimize previously used hormonal treatments to achieve shorter induction treatments, longer spermiation periods and/or higher sperm quality. Fish treated for just 3 weeks (treatment E) or until the onset of spermiation (treatment C) showed the worst results, while the treatment consisting of weekly administration of 1.5 IU hCG g−1 fish (treatment A) induced the highest percentage of spermiating males, the highest number of sperm samples and sperm volumes and densities similar to the rest of the treatments (B: half hormone dosage, or D: biweekly administration). Evaluation of the sperm quality was performed by computer-assisted sperm analysis (CASA), considering the percentage of total motile spermatozoa, the percentage of fast and medium-velocity spermatozoa, as well as different motility parameters. Sperm samples from A-D groups showed between 44% and 54% motile spermatozoa, and between 10% and 15% fast spermatozoa, while samples from E-treated males showed 0% motile cells. No significant differences were found in the spermatozoa straight line velocity (VSL), curvilinear velocity (VCL) or the angular velocity (VAP), neither spermatozoa beating cross frequency (BCF) between A–D groups.

The high sperm density, together with the short spermatozoa swimming time, makes European eel sperm manipulation and assessment for quality difficult. Two diluting media (K15 and K30) previously designed for Japanese eel sperm were tested. After 24 h, European eel sperm showed significant reduction in the percentage of motile spermatozoa after activation and different motility parameters (VAP, angular velocity; VCL, curvilinear velocity; VSL, straight line velocity; BCF, beating cross frequency), concluding that these media are not suitable to preserve the sperm of this species. After a hormonal treatment to induce spermiation, sperm volume, density and motility were recorded at weekly samplings. The variation of the osmolality (325–330 mOsm kg−1), pH (8.4–8.6) and the ionic composition (concentration of Na+, K+, Mg2+ and Ca2+) of the seminal plasma were registered. Physio-chemical results were related with sperm quality throughout the treatment, to determine which must be the suitable characteristics of one extender for the sperm of this species, and to find the best conditions to obtain suitable cryopreservation media for European eel sperm. K+ concentration increased, while Ca2+ and Mg2+ concentrations showed a progressive reduction in correlation with the sperm quality improvement. Na+ showed a decreasing, but not significant tendency. P1 and P2 freezing media were designed considering the physio-chemical parameters as well as the ionic composition shown by the best quality sperm samples, and then compared with the previously described solutions, TNK and K30. Sperm quality was determined, checking the percentage of motile spermatozoa and motility parameters using computer-assisted sperm analysis (CASA) software. Samples were frozen after dilution (1:5, 1:20, 1:100) in different freezing media supplemented with 10% dimethyl sulfoxide (DMSO). After thawing, samples frozen with low dilution ratio (1:5) in TNK and P1 media showed higher, although not significant, spermatozoa survival (35.5 ± 14.5 and 36.6 ± 6.7%). The addition of l-α-phosphatidylcholine to the media seems to have a positive effect, as reported in the Japanese eel.

[EN] This study looks at the correlations that fatty acids have with different tissues in the European eel (Anguilla anguilla L.) during hormonally-induced sexual maturation, with different sperm quality parameters. In order to evaluate the different dynamics of the use of fatty acids, a categorization of the results from each sperm quality parameter (volume, concentration, motility and velocity) was performed. Low and moderate correlations were observed between muscle tissue and some sperm quality parameters but no high correlations were found. Eicosapentaenoic add (20:5n3, EPA) in the liver seems to have a role in determining the volume of sperm produced. This can be explained by the fact that EPA is a major requirement in the early phases of sperm production (probably as a component of the spermatozoal membrane). In addition, the levels of alpha-linolenic acid (18:3-n3, ALA) and linoleic acid (18:2-n6, LA) in the liver decreased when sperm motility increased. In all the tissues, a negative correlation was observed between arachidonic acid (20:4n-6, ARA) and the different sperm velocity parameters. The fact that an increase in the consumption of ARA coincides with an increase in the speed of spermatozoa, highlights the important role that this fatty acid plays not only in sperm production, but also in sperm velocity. All this information could prove useful in the development of suitable broodstock diets to improve sperm quality and subsequently, the larval development of this species. (C) 2014 Elsevier Inc All rights reserved., This work was funded by the European Community's Seventh Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no. 245257 (PRO-EEL), and COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE). Victor Gallego, Ilaria Mazzeo and M. Carmen Vilchez had predoctoral grants from the Spanish Ministry of Science and Innovation (MICINN), Generalitat Valenciana, and UPV PAID Programme (2011-S2-02-6521), respectively. David S. Penaranda was supported by a contract co-financed by MICINN and UPV (PTA2011-4948-I). Rosa Baeza was supported by a contract funded by PRO-EEL. Authors want to thank Vicente Javier Moya Salvador for his technical assistance with gas chromatography analyses.

We examined the effect of female exposure to heatwave during blastocyst formation on their reproductive performance and its effect on transcriptome in blastocyst and endometrial tissue. In this study, a total of 72 rabbit does were artificially inseminated and divided into two environmental groups 2 days later: does under conventional conditions (maintained between 14-22 degrees C, n = 29) and does heat stressed in a climatic chamber (maintained between 32-37 degrees C, n = 43). The heat-stressed group were kept under these conditions for 3 days and returned to conventional conditions thereafter. Five days post-insemination, 48 does were slaughtered to collect blastocyst and endometrium samples. mRNA transcripts from OCT-4, VEGF, erbB3, Ifn-gamma, HSP70 and HSP90 were analysed by qRT-PCR. At day 12 of gestation, 24 females were examined by laparoscopy to evaluate implanted embryos and at birth the total kits born and individual weights were recorded. Results revealed no gene expression changes in blastocyst and endometrial tissue under heatwave exposure. Moreover, our results demonstrated that rabbit embryos developed from 8-16 cells to blastocyst during a heatwave did not affect implantation rates, total number of kits born and foetal losses. In summary, these results demonstrate that heatwave period is not a critical point in the reproductive performance of rabbits during blastocyst formation., This work was supported by the Spanish Research Projects AGL2008-03274 and AGL2011-30170-C02-01. Estrella Jimenez-Trigos and Carmen Naturil-Alfonso were supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2010/262 and ACIF/2013/296, respectively). The English text version was revised by N. Macowan English Language Service.

There has been a marked reduction in natural stocks of eels (genus Anguilla) over the past 60 years, and the culture of eels is still based on the capture of very large quantities of juveniles. It is necessary to close the life cycle in captivity in order to ease the pressure on wild populations. The aims of the present study were to evaluate sperm subpopulations (through cluster analysis of computer-aided sperm analysis data) in the European eel (Anguilla anguilla) and to assess the effects of motility acquisition time after activation (i.e. at 30, 60 and 90s), the thermal regimen (i.e. 108C (T10) or 158C (T15) and up to 208C, or constant at 208C (T20)) and hormonal treatments (i.e. human chorionic gonadotropin (hCG), recombinant (r) hCG or pregnant mare serum gonadotropin (PMSG)) on these subpopulations. In all cases, we obtained three subpopulations of spermatozoa: low velocity and linear (S1); high velocity with low linearity (S2); and high velocity and linear (S3; considered high quality). Total motility and S1 were affected by acquisition time; thus, 30s is recommended as the standard time for motility acquisition. When eels were kept at 208C (T20), motility data fitted quadratic models, with the highest motility and proportion of S3 between Weeks 8 and 12 after the first injection. Lower temperatures (T10,T15) delayed spermiationand the obtainingof high-quality spermatozoa (S3),but did notseem to alter the spermiation process (similar subpopulation pattern). Conversely, the hormonal treatments altered both the dynamics of the subpopulation pattern and the onset of spermiation (with PMSG delaying it). Total motility and the yield of S3 with the widely used hCG treatment varied throughout the spermiation period. However, using rhCG allowed us to obtain high-quality and constant motility for most of the study (Weeks 7-20), and the S3 yield was also higher overall (61.8 � 1.3%; means.e.m.) and more stable over time than the other hormonal treatments (averaging 53.0 � 1.4%). Using T20 and rhCG would be more economical and practical, allowing us to obtain a higher number of S3 spermatozoa over an extended time. Additional keywords: computer-aided sperm analysis, European eel, motility activation.

Studies of embryo cryopreservation efficiency have focused mainly on technical and embryo factors. To determine how a slow freezing process affects embryo and fetal development, we studied in vivo development ability after the freezing procedure by assessing blastocyst development at Day 6, implantation, and birth rates. A transcriptional microarray study was also performed to compare gene expression of 6-day-old rabbit embryos previously frozen and transferred into recipient rabbit females to their in vivo counterparts. Our goal was to study which alteration caused by the freezing procedure still remained in late blastocyst stage just at the time when the implantation process began. A microarray specifically designed to study rabbit gene expression profiling was used in this study. Lower implantation and birth rates were obtained in frozen embryos than in the control group (29.9% and 25.7% vs 88.5% and 70.8% for frozen and control embryos, respectively). Likewise, differences were also observed in gene expression profiles. Compared to 6-day-old in vivo-derived embryos, viable frozen embryos presented 70 differentially expressed genes, 24 upregulated and 46 downregulated. In conclusion, our findings showed that the slow freezing process affected late blastocyst development, implantation, and birth rates and that the gene expression alterations identified at late blastocyst stage could be useful in understanding the differences in developmental potential observed and the deficiencies that might hinder implantation and fetal development.

Studies of embryo cryopreservation efficiency have focused mainly on technical and embryo factors. To determine how a slow freezing process affects embryo and fetal development, we studied in vivo development ability after the freezing procedure by assessing blastocyst development at Day 6, implantation, and birth rates. A transcriptional microarray study was also performed to compare gene expression of 6-day-old rabbit embryos previously frozen and transferred into recipient rabbit females to their in vivo counterparts. Our goal was to study which alteration caused by the freezing procedure still remained in late blastocyst stage just at the time when the implantation process began. A microarray specifically designed to study rabbit gene expression profiling was used in this study. Lower implantation and birth rates were obtained in frozen embryos than in the control group (29.9% and 25.7% vs 88.5% and 70.8% for frozen and control embryos, respectively). Likewise, differences were also observed in gene expression profiles. Compared to 6-day-old in vivo-derived embryos, viable frozen embryos presented 70 differentially expressed genes, 24 upregulated and 46 downregulated. In conclusion, our findings showed that the slow freezing process affected late blastocyst development, implantation, and birth rates and that the gene expression alterations identified at late blastocyst stage could be useful in understanding the differences in developmental potential observed and the deficiencies that might hinder implantation and fetal development.

Sperm production has been obtained from European and Japanese eels, but its quality and quantity tend to be changeable. So, its cryopreservation has been tried in both species. Dimethyl sulfoxide (Me(2)SO) is the best cryoprotectant for European eel sperm, but increases the medium osmolality, inducing the activation of spermatozoa motility. To avoid this, different combinations of pH (6.5 and 8.5) and NaHCO(3) concentrations (20, 40 and 80mM) were tested with two Me(2)SO concentrations (5% and 10%). Foetal bovine serum (FBS, 25%v/v) was added as a membrane protector to all the freezing media used in the different experiments. The highest Me(2)SO and NaHCO(3) concentrations at pH 6.5 caused the best post-thawing motility (26+/-4%). A second experiment was carried out testing media with Me(2)SO 10% with additional NaHCO(3) concentrations (100 and 120 mM). The highest post-thawing motility (38+/-3%) was found in the media containing NaHCO(3) 100mM, but no significant difference was observed compared with the best in the previous experiment (NaHCO(3) 80 mM). In a parallel experiment, aiming to improve the protection against the cryopreservation process, bovine serum albumin (BSA, 5%w/v) was added instead of FBS. Lower motilities were registered with BSA as membrane protector. Spermatozoa activation caused by addition of Me(2)SO can be prevented using high NaHCO(3) concentrations, improving the cryopreservation process. This effect seems be based on some of the products dissociated from NaHCO(3) in aqueous solution, affecting the intracellular pH, essential in the sperm motility.