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Simple Summary A reduction in fishmeal in diets is essential to achieve the aim of sustainable production. In the current work, using a plant protein blend of wheat gluten, wheat and soybean meal supplemented with Tau, Val, Lys and Met, a 10% higher fishmeal substitution without affecting growth and health parameters has been accomplished. Abstract The aquaculture of carnivorous fish is in continuous expansion, which leads to the need to reduce the dependence on fishmeal (FM). Plant proteins (PP) represent a suitable protein alternative to FM and are increasingly used in fish feed. However, PP may lead to stunted growth and enteritis. In the current study, the effect of high FM substitution by PP sources on the growth, mortality and intestinal health of rainbow trout (Oncorhynchus mykiss) was evaluated in terms of the histological intestine parameters and expression of genes related to inflammation (IL-1β, IL-8 and TGF-β) and immune responses (Transferrin, IgT and IFN-γ). The results show that a total substitution registered lower growth and survival rates, probably due to a disruption to the animal’s health. Confirming this hypothesis, fish fed FM0 showed histological changes in the intestine and gene changes related to inflammatory responses, which in the long-term could have triggered an immunosuppression. The FM10 diet presented not only a similar expression to FM20 (control diet), but also similar growth and survival. Therefore, 90% of FM substitution was demonstrated as being feasible in this species using a PP blend of wheat gluten (WG) and soybean meal (SBM) as a protein source.

[EN] BackgroundThe impossibility of closing the life cycle of the European eel (Anguilla anguilla) in captivity troubles the future of this critically endangered species. In addition, the European eel is a highly valued and demanded resource, thus the successful closing of its life cycle would have a substantial economic and ecological impact. With the aim of obtaining the highest gamete quality, the study of the effects of environmental factors, such as temperature, on reproductive performance may prove valuable. This is especially true for the exposure to cold water, which has been reported to improve sexual development in multiple other Actinopterygii species.ResultsEuropean eel males treated with cold seawater (10 degrees C, T10) for 2weeks showed an increase in the proliferation and differentiation of spermatogonial cells until the differentiated spermatogonial type A cell stage, and elevated testosterone and 11-ketotestosterone plasma levels. Transcriptomes from the tissues of the brain-pituitary-gonad (BPG) axis of T10 samples revealed a differential gene expression profile compared to the other experimental groups, with clustering in a principal component analysis and in heat maps of all differentially expressed genes. Furthermore, a functional analysis of differentially expressed genes revealed enriched gene ontology terms involved in the regulation of circadian rhythm, histone modification, meiotic nuclear division, and others.ConclusionsCold seawater treatment had a clear effect on the activity of the BPG-axis of European eel males. In particular, our cold seawater treatment induces the synchronization and increased proliferation and differentiation of specific spermatogonial cells. In the transcriptomic results, genes related to thermoception were observed. This thermoception may have caused the observed effects through epigenetic mechanisms, since all analysed tissues further revealed differentially expressed genes involved in histone modification. The presented results support our hypothesis that a low temperature seawater treatment induces an early sexual developmental stage in European eels. This hypothesis is logical given that the average temperature experienced by eels in the early stages of their oceanic reproductive migration is highly similar to that of this cold seawater treatment. Further studies are needed to test whether a cold seawater treatment can improve the response of European eels to artificial hormonal treatment, as the results suggest., This work was subsidized by the Spanish Ministry of Science and Innovation (REPRO-TEMP project, AGL2013-41646-R), the Spanish Ministry of Innovation, Science and Universities (EELGONIA project, RTI2018-096413-B-I00), and by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS), including the CR and JGHJ predoctoral contracts. JGHJ was granted a Short Term Scientific Mission by the COST Office (COST Action AQUAGAMETE: 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). The funding bodies had no role in the study, analysis, interpretation of data, or in writing the manuscript.

[EN] 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., This publication was funded by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS), the COST Office (COST Action FA 1205, AQUAGAMETE), and the Research Centre of Excellence -1476-4/2016/FEKUT

[EN] Activation at fertilization of the vertebrate egg is triggered by Ca2+ waves. Recent studies suggest the phospholipase C zeta (PLC zeta), a sperm-specific protein, triggers egg activation by an 1P3-mediated Ca2+ release and allow Ca2+ waves at fertilization. In the present study we cloned, characterized, and phylogenetically positioned the European eel PLC zeta (PLC zeta 1). 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 PLC zeta 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. plc zeta was expressed in brain of male and female, in testis but not in ovaries. By first time in vertebrates, it is reported plc zeta 1 expression in the pituitary gland. Testis plc zeta 1 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 PLC zeta 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. (C) 2015 Elsevier Inc. All rights reserved., Funded by the SPERMOT project (Spanish Ministry of Science and Innovation, MICINN; AGL2010-16009). M.C. Vílchez has a predoctoral grant from UPV PAID Programme (2011-S2-02-6521), Marina Morini has a predoctoral grant from Generalitat Valenciana (Programa Grisolía, GRISOLIA/2012/006), Victor Gallego has a postdoctoral grant (UPV; PAID-10-14), and David S. Peñaranda was a contract cofinanced by MICINN and UPV (PTA2011-4948-I). Grants to attend meetings from COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE).

[EN] 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 90 s), the thermal regimen (i.e. 10 degrees C (T10) or 15 degrees C (T15) and up to 20 degrees C, or constant at 20 degrees C (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, 30 s is recommended as the standard time for motility acquisition. When eels were kept at 20 degrees C (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 spermiation and the obtaining of high-quality spermatozoa (S3), but did not seem 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%; mean +/- s.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., This study was funded by the European Community's 7th Framework Program under the Theme 2 'Food, Agriculture and Fisheries, and Bio-technology', grant agreement no. 245257 (PRO-EEL) and Generalitat Valenciana (ACOMP/2012/086). VG and MCV have predoctoral grants from the Spanish Ministry of Economy and Competitiveness (AGL2010-16009) and Universitat Politecnica de Valencia (UPV) PAID Program (2011-S2-02-6521), respectively. DSP was supported by a contract cofinanced by Ministry of Science and Innovation (MICINN) and UPV (PTA2011-4948-I). FM-P was supported by the Ramon y Cajal program (MICINN, RYC-2008-02560).

[EN] 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 measurements 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 facilitated 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 increase 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, This study was 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). Ilaria Mazzeo and Victor Gallego had predoctoral grants from Generalitat Valenciana and Spanish Ministry of Science and Innovation (MICINN), respectively. David S. Penaranda has a postdoc grant from UPV (CEI-01-10), mobility grants from UPV (PAID-00-11) and the Research Council of Norway (EJ/hsm IS-SIP, 2009) and also has been supported by a contract co-financed by MICINN and UPV (PTA2011-4948-I). F.-A. Weltzien received funding from the Norwegian University of Life Sciences.

[EN] Contents 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., This work was supported by Generalitat Valenciana research programme (Prometeo 2009/125). The authors thank Neil Macowan Language Services for revising the English version of the manuscript.

[EN] 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 6days of gestation) and gestational (at 12days 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 6days of gestation, related with embryo development and implantation process, such as Oct-4, epidermal growth factor receptor 3 (erbB3), Transforming Growth Factor ß2, Vascular Endothelial Growth Factor and Interferon ¿ 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 12days 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 6days 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. © 2011 Blackwell Verlag GmbH., We are grateful to the Researcher Personal Training Programme from Valencia Polytechnic University. This work was supported by the Spanish Research Project (CICYT AGL2008-03274). The authors thank Neil Macowan Language Services for revising the English version of the manuscript.

[EN] Contents 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., We are grateful to the Researcher Personal Training Programme from Valencia Polytechnic University. This work was supported by the Spanish Research Project (CICYT AGL2008-03274) and Generalitat Valenciana Research Programme (Prometeo 2009/125). The authors thank Neil Macowan Language Services for revising the English version of the manuscript.