David S. Peñaranda

Molecular and physiological study of the artificial maturation process in European eel males: From brain to testis

European eel males can be artificially matured (1.5IU hCG/g fish), but the regulatory mechanisms of their reproductive development are practically unknown. Spermatogenic stages (S1-S6), biometric characters [eye index (EI), gonadosomatic index (GSI), hepatosomatic index (HSI)] and sperm quality parameters (motility, viability and head spermatozoa morphometry) were analysed. Moreover, the present study evaluated the expression of GnRHs (mammal and chicken II Gonadotropin Release Hormone I) and gonadotrophins (FSHbeta and LHbeta) during hormonal treatment, as well as 11-ketotestosterone (11-KT) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) plasma levels. One week was enough to observe the S2 of gonad development, but it was necessary to reach the 7th week of treatment to obtain animals that presented the most advanced stage of development (S6). Differential regulation of the two GnRH expressions was found, supporting the main role of mGnRH in the control of gonadotrophin release. One hCG injection was enough to dramatically decrease the FSHbeta expression, being close to zero during the rest of the treatment. LHbeta expression and 17,20beta-P registered a significant increase in the same stage of development, S3/4, confirming the role of this gonadotrophin in the last steps of maturation and 17,20beta-P in the spermatozoa maturation. The 11-KT increased with GSI, and the highest 11-KT values coincided with the advanced steps of spermatogenesis prior to spermiation. Being consistent with the known role of the steroid in these processes. Furthermore, this study supports a role for 11-KT in stimulating eye growth, presenting high values when EI increased. Sperm production was obtained from the 4th week of treatment, but it was in the 8th week when a significant increase was observed in sperm quality [viability, high motility (>75%)].

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

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.

Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel (Anguilla anguilla) females

We examined the effect of temperature in European silver eels during their maturation induced by injections of carp pituitary extract on endocrine parameters: pituitary fshβ and lhβ expression, plasma 17β-estradiol (E2) and vitellogenin, estrogen receptor 1 (esr1), and vitellogenin 2 (vtg2) expression in liver. A variable thermal regime (T10) that increased from 10° to 14° and 17°C was compared with a constant 20°C regime (T20) during 12 weeks. T10 caused a faster development until week 8, higher fshβ, lhβ, esr1 expression, and higher E2 levels. The results strongly suggest that T10 is inducing a higher endogenous FSH level which increases the E2 circulating level during vitellogenesis. A variable thermal regime induced an fshβ expression and E2 profile in vitellogenic hormonally matured eel females that were more similar to the profile observed in other naturally maturing fish.

Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software

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, 10× 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 cameras 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.

Improvement of European eel sperm cryopreservation method by preventing spermatozoa movement activation caused by cryoprotectants.

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.

The use of agricultural substrates to improve methane yield in anaerobic co-digestion with pig slurry: effect of substrate type and inclusion level.

Anaerobic co-digestion of pig slurry with four agricultural substrates (tomato, pepper, persimmon and peach) was investigated. Each agricultural substrate was tested in co-digestion with pig slurry at four inclusion levels: 0%, 15%, 30% and 50%. Inclusion levels consisted in the replacement of the volatile solids (VS) from the pig slurry with the VS from the agricultural substrate. The effect of substrate type and inclusion level on the biochemical methane potential (BMP) was evaluated in a batch assay performed at 35 °C for 100 days. Agricultural substrates chemical composition was also analyzed and related with BMP. Additionally, Bacteria and Archaea domains together with the four main methanogenic archaeal orders were quantified using quantitative real-time TaqMan polymerase chain reaction (qPCR) at the end of the experiment to determine the influence of agricultural substrate on sludges microbial composition. Results showed that vegetable substrates (pepper and tomato) had higher lipid and protein content and lower carbohydrates than fruit substrates (persimmon and peach). Among substrates, vegetable substrates showed higher BMP than fruit substrates. Higher BMP values were obtained with increasing addition of agricultural substrate. The replacement of 50% of VS from pig slurry by tomato and pepper increased BMP in 41% and 44%, respectively compared with pig slurry only. Lower increments in BMP were achieved with lower inclusion levels. Results from qPCR showed that total bacteria and total archaea gene concentrations were similar in all combinations tested. Methanomicrobiales gene concentrations dominated over the rest of individual archaeal orders.

Effects of Slow Freezing Procedure on Late Blastocyst Gene Expression and Survival Rate in Rabbit

ABSTRACT 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.

Effects of pH, Sodium Bicarbonate, Cryoprotectants and Foetal Bovine Serum on the Cryopreservation of European Eel Sperm

The main objective of the present study was to evaluate the influence of pH and bicarbonate concentration in the activation or inhibition of European eel (Anguilla anguilla) spermatozoa and to evaluate the effect of different cryoprotectants: dimethyl sulphoxide (DMSO), acetamide, ethylene glycol, propanol, glycerol and methanol (MeOH). The effect of these factors was evaluated comparing the percentage of motile cells, the percentage of alive cells (by Hoechst staining) and the spermatozoa morphometry pre- and post-cryopreservation (by computer-assisted morphology analysis). Based on the above findings, three cryoprotectants (DMSO, MeOH and glycerol) were chosen and evaluated in two media (P1 and P1 modified) with different concentrations of NaHCO(3) and in the presence or absence of foetal bovine serum (FBS). The effect of these factors was evaluated comparing the percentage of alive and motile cells post-cryopreservation. DMSO was the cryoprotectant showing better results in relation to the percentage of spermatic alive cells post-freezing and caused a smaller modification of the head spermatozoa morphology. The combination of P1-modified medium with DMSO and containing FBS increased slightly but significantly the percentage of motile spermatozoa post-cryopreservation.

Subpopulation pattern of eel spermatozoa is affected by post-activation time, hormonal treatment and the thermal regimen

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. 10°C (T10) or 15°C (T15) and up to 20°C, or constant at 20°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, 30s is recommended as the standard time for motility acquisition. When eels were kept at 20°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.

Effect of Embryonic Genotype on Reference Gene Selection for RT‐qPCR Normalization

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.