Long-Term Phenotypic and Proteomic Changes Following Vitrified Embryo Transfer in the Rabbit Model

[EN] This study was conducted to demonstrate how a vitrified embryo transfer procedure incurs phenotypic and molecular changes throughout life. This study reports the first evidence describing that embryonic manipulation during a vitrified embryo transfer cycle induced molecular modifications, concerning oxidative phosphorylation and dysregulations in zinc and lipid metabolism in liver tissue, which has been reported as responsible for postnatal variations of the phenotype. Nowadays, assisted reproductive technologies (ARTs) are considered valuable contributors to our past, but a future without their use is inconceivable. However, in recent years, several studies have evidenced a potential impact of ART on long-term development in mammal species. To date, the long-term follow-up data are still limited. So far, studies have mainly focused on in vitro fertilization or in vitro culture, with information from gametes/embryos cryopreservation field being practically missing. Herein, we report an approach to determine whether a vitrified embryo transfer procedure would have long-term consequences on the offspring. Using the rabbit as a model, we compared animals derived from vitrified-transferred embryos versus those naturally conceived, studying the growth performance, plus the weight throughout life, and the internal organs/tissues phenotype. The healthy status was assessed over the hematological and biochemical parameters in peripheral blood. Additionally, a comparative proteomic analysis was conducted in the liver tissue to investigate molecular cues related to vitrified embryo transfer in an adult tissue. After vitrified embryo transfer, birth weight was increased, and the growth performance was diminished in a sex-specific manner. In addition, vitrified-transferred animals showed significantly lower body, liver and heart weights in adulthood. Molecular analyses revealed that vitrified embryo transfer triggers reprogramming of the liver proteome. Functional analysis of the differentially expressed proteins showed changes in relation to oxidative phosphorylation and dysregulations in the zinc and lipid metabolism, which has been reported as possible causes of a disturbed growth pattern. Therefore, we conclude that vitrified embryo transfer is not a neutral procedure, and it incurs long-term effects in the offspring both at phenotypic and molecular levels. These results described a striking example of the developmental plasticity exhibited by the mammalian embryo.Funding from the Ministry of Economy, Industry and Competitiveness (Research project: AGL2017-85162-C2-1-R and AGL2014-53405-C2-1-P) is acknowledged. X.G.D. was supported by a research grant from the Ministry of Economy, Industry and Competitiveness (BES-2015-072429).Garcia-Dominguez, X.; Marco-Jiménez, F.; Peñaranda, D.; Vicente Antón, JS. (2020). Long-Term Phenotypic and Proteomic Changes Following Vitrified Embryo Transfer in the Rabbit Model. 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Feed restriction regime in a rabbit line selected for growth rate alters oocyte maturation manifested by alteration in msy2 gene expression

[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).Naturil Alfonso, C.; Peñaranda, D.; Vicente Antón, JS.; Marco-Jiménez, F. (2017). 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Successful inclusion of high vegetable protein sources in feed for rainbow trout without decrement in intestinal health

[EN] 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. 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-1 beta, IL-8 and TGF-beta) and immune responses (Transferrin, IgT and IFN-gamma). 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.Vélez-Calabria, G.; Peñaranda, D.; Jover Cerda, M.; Martínez-Llorens, S.; Tomas-Vidal, A. (2021). Successful inclusion of high vegetable protein sources in feed for rainbow trout without decrement in intestinal health. Animals. 11(12):1-18. https://doi.org/10.3390/ani11123577S118111

Effects of Eco-Organic Feed on Growth Performance, Biometric Indices, and Nutrient Retention of Gilthead Seabream (Sparus aurata)

[EN] This study examined how eco-organic feed affects the growth performance, nutrient efficiency, feed utilisation, and body composition of gilthead seabream. Six different diets were tested, including a control diet (CONT) without organic ingredients and four diets with 100% organic ingredients: trout (TRO), seabass (SBS), poultry (POU), and mix (MIX), along with a control organic diet (ORG) containing organic ingredients and 30% fishmeal. The experiment lasted 70 days, and the fish were fed twice a day, starting with an initial weight of 60.5 g. The results showed that the highest growth rates were observed in fish fed the ORG and CONT diets containing fishmeal. Conversely, the POU diet resulted in the lowest growth rate, survival rate, and highest value for feed conversion ratio (FCR). Almost all essential amino acid efficiency values were high in fish fed the ORG and CONT diets. Still, significant differences were noted in the retention efficiency of fatty acids across all diets. The retention efficiency was higher in the CONT diet, followed by the ORG diet. However, the economic conversion rate was lower for CONT, SBS, TRO, and MIX. Overall, using organic diets of animal origin impacted the growth performance of gilthead seabream, but it is still a promising approach.This project had been developed with the collaboration of the Biodiversity Foundation (Spanish Ministry for Ecological Transition and the Demographic Challenge), through the Pleamar Program, co-financed by the European Maritime and Fisheries Fund (EMFF). A full scholarship from the Ministry of Higher Education of the Arab Republic of Egypt funds the researcher Eslam TefalTefal, E.; Tomas-Vidal, A.; Martínez-Llorens, S.; Jauralde García, I.; Peñaranda, D.; Jover Cerda, M. (2023). Effects of Eco-Organic Feed on Growth Performance, Biometric Indices, and Nutrient Retention of Gilthead Seabream (Sparus aurata). Sustainability. 15(14):1-16. https://doi.org/10.3390/su151410750116151

Intestinal Explant Cultures from Gilthead Seabream (Sparus aurata, L.) Allowed the Determination of Mucosal Sensitivity to Bacterial Pathogens and the Impact of a Plant Protein Diet

[EN] 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 1 beta (IL1-beta), interleukin 6 (IL-6) and cyclooxygenase 2(COX-2).The research was supported by a grant financed by the Spanish Ministerio de Economia y Competitividad AGL2015-70487-P. and Generalitat Valenciana, IDIFEDER/2020/029 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. It was additionally granted by 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.Peñaranda, D.; Bäuerl, C.; Tomas-Vidal, A.; Jover Cerda, M.; Estruch, G.; Pérez Martínez, G.; Martínez-Llorens, S. (2020). Intestinal Explant Cultures from Gilthead Seabream (Sparus aurata, L.) Allowed the Determination of Mucosal Sensitivity to Bacterial Pathogens and the Impact of a Plant Protein Diet. International Journal of Molecular Sciences. 21(20):1-20. https://doi.org/10.3390/ijms21207584S1202120Minghetti, M., Drieschner, C., Bramaz, N., Schug, H., & Schirmer, K. (2017). A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC. Cell Biology and Toxicology, 33(6), 539-555. doi:10.1007/s10565-017-9385-xGómez, G. D., & Balcázar, J. L. (2008). 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Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software

[EN] The development of powerful computer-assisted sperm analysis software has made kinetic studies of spermatozoa possible. This system has been used and validated for several species, but some technical questions have emerged regarding fish sample evaluations (i.e., frame rate, sperm dilution, chamber model, time of analysis, magnification lens, etc.). In the present study, we have evaluated the effects of different procedural and biological settings with the aim to correctly measure sperm quality parameters of the European eel. The use of different chambers did not affect the sperm motility parameters. However, regarding lens magnification, 10x was the most accurate lens, showing the least variation in the acquired data. Similarly, the frame rate setting resulted in a dramatic effect in some sperm kinetic parameters, primarily in terms of curvilinear velocity; we therefore recommend using the camera's highest available frame rate setting. Finally, the reduction in sperm motility over postactivation times suggests that sperm analysis should be performed within the first 60 seconds after activation of the European eel sperm. In conclusion, some protocol variables of sperm analysis by computer-assisted sperm analysis software can affect the measurement of eel sperm quality parameters, and should be considered before directly comparing results obtained by different laboratories. Moreover, because marine fish species show relatively similar features of sperm kinetic parameters, these results could be considered in the evaluation of the motility of sperm from other fish species. (C) 2013 Elsevier Inc. All rights reserved.This study was funded from the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology," grant agreement 245257 (Pro-Eel), and Generalitat Valenciana (ACOMP/2011/229). D.S.P. and P.C.F.C. have postdoctoral grants from UPV (CE-01-10) and PAC-EMBRAPA, respectively. I.M. and V.G. have predoctoral grants from Generalitat Valenciana and Spanish MICINN, respectively. The authors thank the Proiser R&D, S.L. team, who performed the task of fractioning the original sequences for the experiment described in section 3.2.Gallego Albiach, V.; Carneiro, PCF.; Mazzeo, I.; Vilchez Olivencia, MC.; Peñaranda, D.; Soler, C.; Pérez Igualada, LM.... (2013). Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software. Theriogenology. 79(7):1034-1040. https://doi.org/10.1016/j.theriogenology.2013.01.019S1034104079

Impact of high dietary plant protein with or without marine ingredients in gut mucosa proteome of gilthead seabream (Sparus aurata, L.)

[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.Estruch, G.; Martínez-Llorens, S.; Tomas-Vidal, A.; Monge-Ortiz, R.; Jover Cerda, M.; Brown, PB.; Peñaranda, D. (2020). Impact of high dietary plant protein with or without marine ingredients in gut mucosa proteome of gilthead seabream (Sparus aurata, L.). Journal of Proteomics. 216:1-13. https://doi.org/10.1016/j.jprot.2020.103672S113216Martínez-Llorens, S., Moñino, A. V., Tomás Vidal, A., Salvador, V. J. M., Pla Torres, M., & Jover Cerdá, M. (2007). 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Intracellular changes in Ca2+, K+ and pH after sperm motility activation in the European eel (Anguilla anguilla): Preliminary results

[EN] Although it is widely accepted that osmolality and ion fluxes are the main factors triggering sperm motility in fish, a complex universal mechanism for sperm motility activation does not exist in fish, and studies of marine fish species are even more scarce. Therefore, the main goal of this study was to estimate the intracellular variations in the main ions involved in sperm activation for the first time in European eel, in order to provide additional new data about this little-known process. It was observed that levels of intracellular Ca2+ and K+ sperm ions increased significantly 30 s after the hyperosmotic shock compared to baseline levels, and remained at this level until 120 s post-activation. In contrast, the intracellular pH remained constant during the first 30 s, and decreased gradually at 60 and 120 s post-activation. Our data agree with the current main theory for explaining motility activation in marine fish, in which internal fluctuations of Ca2+ and K+ seem to participate in sperm activation. In addition, fluorescent images showed that both Ca2+ and K+ were concentrated in the apical area of the sperm head, which corresponds to the location of the eel sperm mitochondria, suggesting this organelle plays an important role in sperm motility activation. (C) 2013 Elsevier B.V. All rights reserved.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 the Spanish Ministry of Science and Innovation (MICINN; AGL2010-16009). Victor Gallego has a predoctoral grant (MICINN; BES-2009-020310) and has been granted a fellowship (EEBB-I-12-05858) of the Spanish Personnel Research Training Programme to carry out this study in the Universidad de Leon (Leon, Spain). Ilaria Mazzeo had a predoctoral grant from GVA. David S. Penaranda has a contract co-financed by MICINN and UPV (PTA2011-4948-I). F. Martinez-Pastor was supported by the Ramon y Cajal program (RYC-2008-02560, MICINN).Gallego Albiach, V.; Martínez Pastor, F.; Mazzeo, I.; Peñaranda, D.; Herraez, P.; Asturiano Nemesio, JF.; Pérez Igualada, LM. (2014). Intracellular changes in Ca2+, K+ and pH after sperm motility activation in the European eel (Anguilla anguilla): Preliminary results. Aquaculture. 418:155-158. https://doi.org/10.1016/j.aquaculture.2013.10.022S15515841

Role of calcium on the initiation of sperm motility in the European eel

[EN] Sperm from European eel males treated with hCG(rec), was washed in a calcium free extender, and sperm motility was activated both in the presence (seawater, SW) and in the absence of calcium (NaCI + EDTA), and treated with calcium inhibitors or modulators. The sperm motility parameters were evaluated by a computer-assisted sperm analysis (CASA) system, and changes in the [Ca2+](i) fluorescence (and in [Na+](i) in some cases) were evaluated by flow cytometry. After sperm motility was activated in a medium containing Ca2+ (seawater, SW) the intracellular fluorescence emitted by Ca2+ increased 4-6-fold compared to the levels in quiescent sperm. However, while sperm activation in a Ca-free media (NaCI + EDTA) resulted in a percentage of motility similar to seawater, the [Ca2+](i) levels did not increase at all. This result strongly suggests that increasing [Ca2+](i) is not a pre-requisite for the induction of sperm motility in European eel sperm. Several sperm velocities (VCL, VSL, VAP) decreased when sperm was activated in the Ca-free activator, thus supporting the theory that Ca2+ has a modulatory effect on sperm motility. The results indicate that a calcium/sodium exchanger (NCX) which is inhibited by bepridil and a calcium calmodulin kinase (inhibited by W-7), are involved in the sperm motility of the European eel. Our results indicate that the increase in [Ca2+](i) concentrations during sperm activation is due to an influx from the external medium, but, unlike in most other species, it does not appear to be necessary for the activation of motility in European eel sperm. (c) 2015 Elsevier Inc All rights reserved.Funded from the SPERMOT project (Spanish Ministry of Science and Innovation, MICINN; AGL2010-16009). M.C. Vilchez has a predoctoral grant from UPV PAID Program (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-I) and UPV (PTA2011-4948-I). Grants to attend meetings were received from COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE).Pérez Igualada, LM.; Vilchez Olivencia, MC.; Gallego Albiach, V.; Morini, M.; Peñaranda, D.; Asturiano Nemesio, JF. (2016). Role of calcium on the initiation of sperm motility in the European eel. Comparative Biochemistry and Physiology - Part A: Molecular and Integrative Physiology. 191:98-106. https://doi.org/10.1016/j.cbpa.2015.10.009S9810619

Impact of Fishmeal Replacement in Diets for Gilthead Sea Bream (Sparus aurata) on the Gastrointestinal Microbiota Determined by Pyrosequencing the 16S rRNA Gene

[EN] Recent studies have demonstrated the impact of diet on microbiota composition, but the essential need for the optimization of production rates and costs forces farms and aquaculture production to carry out continuous dietary tests. In order to understand the effect of total fishmeal replacement by vegetable-based feed in the sea bream (Sparus aurata), the microbial composition of the stomach, foregut, midgut and hindgut was analysed using high-throughput 16S rDNA sequencing, also considering parameters of growth, survival and nutrient utilisation indices.A total of 91,539 16S rRNA filtered-sequences were analysed, with an average number of 3661.56 taxonomically assigned, high-quality sequences per sample. The dominant phyla throughout the whole gastrointestinal tract were Actinobacteria, Protebacteria and Firmicutes. A lower diversity in the stomach in comparison to the other intestinal sections was observed. The microbial composition of the Recirculating Aquaculture System was totally different to that of the sea bream gastrointestinal tract. Total fishmeal replacement had an important impact on microbial profiles but not on diversity. Streptococcus (p-value: 0.043) and Photobacterium (p-value: 0.025) were highly represented in fish fed with fishmeal and vegetable-meal diets, respectively. In the stomach samples with the vegetable diet, reads of chloroplasts and mitochondria from vegetable dietary ingredients were rather abundant. Principal Coordinate Analysis showed a clear differentiation between diets in the microbiota present in the gut, supporting the presence of specific bacterial consortia associated with the diet.Although differences in growth and nutritive parameters were not observed, a negative effect of the vegetable diet on the survival rate was determined. Further studies are required to shed more light on the relationship between the immune system and sea bream gastrointestinal tract microbiota and should consider the modulation of the microbiota to improve the survival rate and nutritive efficacy when using plant-based diets.Project Name: Aquaculture feed without fishmeal(SP20120603). Grant Numbers: 6.000,00. Funding Institution: Vicerrectorat d'Investigacio, Innovacio i Transferencia - Universitat Politecnica de Valencia. Author who received the funding: ATV. URL of funder: http://www.upv.es/entidades/VIIT/info/indexnormalc.html. The first author was supported by a contract-grant (Contrato Pre-doctoral del Programa para la Formacion de Personal Investigador - FPI) from Programa de Ayudas de Investigacion y Desarrollo (PAID) of Universitat Politecnica de Valencia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Estruch, G.; Collado Amores, MC.; Peñaranda, D.; Tomas-Vidal, A.; Jover Cerda, M.; Pérez-Martínez, G.; Martínez-Llorens, S. (2015). Impact of Fishmeal Replacement in Diets for Gilthead Sea Bream (Sparus aurata) on the Gastrointestinal Microbiota Determined by Pyrosequencing the 16S rRNA Gene. PLoS ONE. 10:1-22. doi:10.1371/journal.pone.0136389S1221