Growth, Survival, and Intestinal Health Alterations in Mediterranean Yellowtail (Seriola dumerili) Due to Alternatives to Fishmeal and Fish Oil.

Fishmeal and fish oil substitution in aquafeeds might have adverse effects on fish growth and health, mainly in carnivorous species, such as Mediterranean yellowtail (Seriola dumerili). Mediterranean yellowtail shows great potential as an alternative aquaculture species due to its fast growth and high price on the market, but the need for high-quality protein and fatty acid content in its diets is limiting its production. In order to improve the sustainability of its production, this study was conducted with 360 fish of 35 g to evaluate the effects on fish growth and health. Six diets were used: one control diet without replacement, three with FM replacement (FM66, FM33, and FM0) (33%, 66%, and 100% FM replacement), and two with FO replacement (FO50 and FO0) (50% and 100% FO replacement). The substitution of FM was with vegetable (VM) (corn gluten) and animal (AM) (krill and meat meal) meals. The reductions in FM and FO of up to 33 and 0%, respectively, did not affect the growth and survival of S. dumerili at the intestinal morphology level, except for the anterior intestine regarding the lower villi length and width and the posterior intestine regarding the lower width of the lamina propria. On the other hand, the substitution of fish ingredients in the diet affects liver morphology, indicating alterations in the major diameter of hepatocytes or their nuclei. Finally, diet did not affect the gut microbiota with respect to the control, but significant differences were found in alpha and beta diversity when FO and FM microbiota were compared. A 66% FM replacement and total FO replacement would be possible without causing major alterations in the fish.

Organic Ingredients as Alternative Protein Sources in the Diet of Juvenile Organic Seabass (Dicentrarchus labrax).

The use of organic ingredients as a source of protein in aquaculture diets has gained significant attention due to the growing demand for organic seafood products. This study aimed to evaluate the potential for the use of organic ingredients as protein sources in the diet of juvenile organic seabass (Dicentrarchus labrax). A total of 486 juvenile seabass with an average weight of 90 g were fed six diets containing varied organic proteins. The control group (CON) was fed a diet with conventional fishmeal from sustainable fisheries as the primary protein source. The other five groups were fed diets with different compositions: organic Iberian pig meal byproduct (IB diet), a combination of organic Iberian pig meal byproduct and insect meal (IB-IN diet), a mix of organic Iberian pig meal byproduct and organic rainbow trout meal byproduct (IB-TR diet), a blend of organic rainbow trout meal byproduct and insect meal (TR-IN), and a mixed diet containing all of these protein sources (MIX diet). Over a 125-day feeding trial, growth performance, feed utilisation, feed digestibility, and histological parameters were assessed. The results showed that the fish fed the control diet had the highest final weight and specific growth rate, followed by the fish fed the TR-IN and IB-TR diets. The IB-TR diet had the highest apparent digestibility coefficients (ADCs) for protein, while the TR-IN diet had the lowest. Histological analysis revealed that fish fed the control diet had the largest nucleus diameter and hepatocyte diameter. Use of IN seems to penalise performance in several ways. Fish fed diets containing insect meal grew less, and those diets had lower digestibility. Fish fed the TR and IB diets grew at rates near that of the control, and the feed had acceptable digestibility.

Hepatic, Muscle and Intestinal Oxidative Status and Plasmatic Parameters of Greater Amberjack (Seriola dumerili, Risso, 1810) Fed Diets with Fish Oil Replacement and Probiotic Addition.

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.

Effect of Additives Inclusion in Gilthead Seabream (Sparus aurata L.) Diets on Growth, Enzyme Activity, Digestibility and Gut Histology Fed with Vegetable Meals.

The fishmeal replacement by vegetable meals or other alternative sources, without affecting fish performance and productivity, is one of the principal challenges in aquaculture. The use of hydrolyzed porcine mucosa (HPM) and nucleotide (NT) concentrates, as feed additives in gilthead seabream (Sparus aurata L.) non-fishmeal diets was assessed in order to determine the possible effects on growth, feed efficiency, protein digestion, and gut histology when these were included in a plant-based diet (HPM 1% and 2%, P1 and P2; NT 250 and 500 ppm, N250 and N500), in comparison with two control diets, AA0 (100% plant-protein-based diet) and FM100 (100% fishmeal-protein-based diet). Diets were assayed in triplicate and the growth assay lasted 134 days. Results showed a significant improvement in all groups in terms of final weight and specific growth rate in comparison with the AA0 group. An improvement in the feed conversion ratio and the protein efficiency ratio was also observed when the additives were included in lower percentages (P1 and N250) compared to the FM100 group. Significant differences were found in hepatosomatic index, villi thickness, and goblet cells. Thus, the inclusion of NT and HPM was tested as beneficial for the improvement of efficiency of plant feed in seabream.

Early Embryo Exposure to Assisted Reproductive Manipulation Induced Subtle Changes in Liver Epigenetics with No Apparent Negative Health Consequences in Rabbit.

Embryo manipulation is a requisite step in assisted reproductive technology (ART). Therefore, it is of great necessity to appraise the safety of ART and investigate the long-term effect, including lipid metabolism, on ART-conceived offspring. Augmenting our ART rabbit model to investigate lipid metabolic outcomes in offspring longitudinally, we detected variations in hepatic DNA methylation ART offspring in the F3 generation for embryonic exposure (multiple ovulation, vitrification and embryo transfer). Through adult liver metabolomics and proteomics, we identified changes mainly related to lipid metabolism (e.g., polyunsaturated fatty acids, steroids, steroid hormone). We also found that DNA methylation analysis was linked to changes in lipid metabolism and apoptosis genes. Nevertheless, these differences did not apparently alter the general health status. Thus, our findings suggest that ART is likely to be a player in embryo epigenetic events related to hepatic homeostasis alteration in adulthood.

Estimation of Phosphorus and Nitrogen Waste in Rainbow Trout (Oncorhynchus mykiss, Walbaum, 1792) Diets Including Different Inorganic Phosphorus Sources.

This study was conducted to evaluate the apparent availability and P and N excretion in rainbow trout (Oncorhynchus mykiss) using different inorganic phosphorus sources. With this goal, fish (153 ± 14.1 g) fed four inorganic P sources were assayed: monoammonium phosphate (MAP, NH4H2PO4), monosodium/monocalcium phosphate (SCP-2%, AQphos+, NaH2PO4/Ca(H2PO4)2·H2O in proportion 12/88), monosodium/monocalcium phosphate (SCP-5%, NaH2PO4/Ca(H2PO4)2·H2O in proportion 30/70) and monocalcium phosphate (MCP, Ca(H2PO4)2·H2O). Phosphorus (P) digestibility, in diets that included MAP and SCP-2% as inorganic phosphorus sources, were significantly higher than for SCP-5% and MCP sources. In relation to the P excretion pattern, independent of the diet, a peak at 6 h after feeding was registered, but at different levels depending on inorganic P sources. Fish fed an MAP diet excreted a higher amount of dissolved P in comparison with the rest of the inorganic P sources, although the total P losses were lower in MAP and SCP-2% (33.02% and 28.13, respectively) than in SCP-5% and MCP sources (43.35% and 47.83, respectively). Nitrogen (N) excretion was also studied, and the fish fed an SCP-5% diet provided lower values (15.8%) than MAP (28.0%). When N total wastes were calculated, SCP-2% and SCP-5% showed the lowest values (31.54 and 28.25%, respectively). In conclusion, based on P and N digestibility and excretion, the SCP-2% diet showed the best results from a nutritional and environmental point of view.

Effect of Embryo Vitrification on the Steroid Biosynthesis of Liver Tissue in Rabbit Offspring.

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.

Long-term and transgenerational phenotypic, transcriptional and metabolic effects in rabbit males born following vitrified embryo transfer.

The advent of assisted reproductive technologies (ART) in mammals involved an extraordinary change in the environment where the beginning of a new organism takes place. Under in vitro conditions, in which ART is currently being performed, it likely fails to mimic optimal in vivo conditions. This suboptimal environment could mediate in the natural developmental trajectory of the embryo, inducing lasting effects until later life stages that may be inherited by subsequent generations (transgenerational effects). Therefore, we evaluated the potential transgenerational effects of embryo exposure to the cryopreservation-transfer procedure in a rabbit model on the offspring phenotype, molecular physiology of the liver (transcriptome and metabolome) and reproductive performance during three generations (F1, F2 and F3). The results showed that, compared to naturally-conceived animals (NC group), progeny generated after embryo exposure to the cryopreservation-transfer procedure (VT group) exhibited lower body growth, which incurred lower adult body weight in the F1 (direct effects), F2 (intergenerational effects) and F3 (transgenerational effects) generations. Furthermore, VT animals showed intergenerational effects on heart weight and transgenerational effects on liver weight. The RNA-seq data of liver tissue revealed 642 differentially expressed transcripts (DETs) in VT animals from the F1 generation. Of those, 133 were inherited from the F2 and 120 from the F3 generation. Accordingly, 151, 190 and 159 differentially accumulated metabolites (DAMs) were detected from the F1, F2 and F3, respectively. Moreover, targeted metabolomics analysis demonstrated that transgenerational effects were mostly presented in the non-polar fraction. Functional analysis of molecular data suggests weakened zinc and fatty acid metabolism across the generations, associated with alterations in a complex molecular network affecting global hepatic metabolism that could be associated with the phenotype of VT animals. However, these VT animals showed proper reproductive performance, which verified a functional health status. In conclusion, our results establish the long-term transgenerational effects following a vitrified embryo transfer procedure. We showed that the VT phenotype could be the result of the manifestation of embryonic developmental plasticity in response to the stressful conditions during ART procedures.

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

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.

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

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.

Cold seawater induces early sexual developmental stages in the BPG axis of European eel males.

BACKGROUND: The 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. RESULTS: European eel males treated with cold seawater (10 °C, T10) for 2 weeks 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. CONCLUSIONS: Cold 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.

De novo European eel transcriptome provides insights into the evolutionary history of duplicated genes in teleost lineages.

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.

Long-term feeding with high plant protein based diets in gilthead seabream (Sparus aurata, L.) leads to changes in the inflammatory and immune related gene expression at intestinal level.

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-1ß (il1ß, 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 (il1ß, 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 ß, 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.

Testing cryopreserved European eel sperm for hybridization (A. japonica × A. anguilla).

The objective of this study was to assess impact of cryopreserved European eel sperm and Japanese eel native sperm on early fertilization, hatch, survival, and malformation rates of larvae, as well as develop molecular techniques to distinguish different eel species. Eggs from Japanese eel females (Anguilla japonica) were artificially fertilized with sperm of Japanese eel males and cryopreserved sperm from European eel (A. anguilla, extender was modified Tanaka solution and methanol as cryoprotectant). There were no statistical differences (p > 0.05) among the measured parameters such as fertilization, hatch and survival after 10 days post-hatch rates due to large individual differences. The malformation rate of larvae compared to the hatching rate was higher in cryopreserved groups than in the control indicating that the methodology needs further refinement. Genetic analyses (PCR-RFLP, PCR-HRM) proved a clear result in the detection of paternal contribution in hybridization between the Japanese and the European eel and applied PCR-HRM method is a quick and cost effective tool to identify illegally imported A. anguilla at the glass eel stage, which can be transported from Europe to Asia.

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation.

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.

Nuclear and membrane progestin receptors in the European eel: Characterization and expression in vivo through spermatogenesis.

Characterization of all the progestin receptor genes (PRs) found in the European eel has been performed. There were five membrane PRs (mPRs): mPRα (alpha), mPRAL1 (alpha-like1), mPRAL2 (alpha-like2), mPRγ (gamma), mPRδ (delta) and two nuclear PRs (nPRs or PGRs): pgr1 and pgr2. In silico studies showed that the C and E(F) domains of Pgr are well conserved among vertebrates whereas the A/B domain is not. Phylogeny and synteny analyses suggest that eel duplicated pgr (pgr1 and pgr2) originated from the teleost-specific third whole genome duplication (3R). mPR phylogeny placed three eel mPRs together with the mPRα clade, being termed mPRα, mPRAL1 and mPRAL2, while the other two eel mPRs clustered with mPRγ and mPRδ clades, respectively. The in vivo study showed differential expression patterns along the brain-pituitary-gonad axis. An increase in nPR transcripts was observed in brain (in pgr1) and pituitary (in pgr1 and pgr2) through the spermatogenesis, from the spermatogonia B/spermatocyte stage to the spermiation stage. In the testis, mPRγ, mPRδ and pgr2 transcripts showed the highest levels in testis with A spermatogonia as dominant germ cell, while the highest mPRα, mPRAL1 and mPRAL2 transcripts were observed in testis from spermiating males, where the dominant germ cell were spermatozoa. Further studies should elucidate the role of both nuclear and membrane progestin receptors on eel spermatogenesis.

Development of sperm vitrification protocols for freshwater fish (Eurasian perch, Perca fluviatilis) and marine fish (European eel, Anguilla anguilla).

Vitrification was successfully applied to the sperm of two fish species, the freshwater Eurasian perch (Perca fluviatilis) and marine European eel (Anguilla anguilla). Sperm was collected, diluted in species-specific non-activating media and cryoprotectants and vitrified by plunging directly into liquid nitrogen without pre-cooling in its vapor. Progressive motility of fresh and vitrified-thawed sperm was evaluated with computer-assisted sperm analysis (CASA). Additional sperm quality parameters such as sperm head morphometry parameters (in case of European eel) and fertilizing capacity (in case of Eurasian perch) were carried out to test the effectiveness of vitrification. The vitrification method for Eurasian perch sperm resulting the highest post-thaw motility (14±1.6%) was as follows: 1:5 dilution ratio, Tanaka extender, 30% cryoprotectant (15% methanol+15% propylene-glycol), cooling device: Cryotop, 2µl droplets, and for European eel sperm: dilution ratio 1:1, with 40% cryoprotectant (20% MeOH and 20% PG), and 10% FBS, cooling device: Cryotop, with 2µl of sperm suspension. Viable embryos were produced by fertilization with vitrified Eurasian perch sperm (neurulation: 2.54±1.67%). According to the ASMA analysis, no significant decrease in head area and perimeter of vitrified European eel spermatozoa were found when compared to fresh spermatozoa.

Role of potassium and pH on the initiation of sperm motility in the European eel.

The role of potassium from the seminal plasma and/or the activation media was examined by selectively removing K+ from this media, and by testing the use of K+ 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 pHi in the quiescent stage was also induced. Intracellular pH (pHi) 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 pHi (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.

The expression of nuclear and membrane estrogen receptors in the European eel throughout spermatogenesis.

Estradiol (E2) 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 E2 signalling pathway along the BPG axis. Stimulation of sexual maturation by weekly injections of human chorionic gonadotropin (hCG) induced a progressive decrease in E2 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 E2. 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.