Oocyte quality and in vivo embryo survival after ovarian stimulation in nulliparous and multiparous rabbit does.

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

Using specific recombinant gonadotropins to induce spermatogenesis and spermiation in the European eel (Anguilla anguilla).

New specific European eel (Anguilla anguilla) recombinant gonadotropins (aarGths) produced in the ovarian cells of Chinese hamsters (CHO) were used to induce maturation in captive male eels. In the first experiment, five different hormonal treatments were assayed: one group was given a constant dose of recombinant European eel follicle-stimulating hormone (aarFsh; 4 µg/fish) for 9 weeks, and the second group received a constant dose of recombinant European eel luteinizing hormone (aarLh; 2 µg/fish) also for 9 weeks. The other three groups were injected with different combinations of both aarGths (some doses constant, some variable). All five treatments stimulated androgen synthesis, but the increase was more pronounced in the fish treated with a combination of both aarGths. Unlike aarLh, aarFsh alone was able to induce spermiation, the best results were achieved in the fish that were treated with a constant dose of aarFSH and an increasing dose of aarLH, with spermiation being induced (20% motile cells) despite the fact that these fish were immature at the start of the experiment. In order to improve sperm quality, a second experiment was performed. Immature males received three constant doses of aarFsh (2.8, 1.4 or 0.7 µg/fish) and increasing doses of aarLh (every 3 weeks; 1, 2, 6 µg/fish). All the treatments induced spermiation, however the best sperm quality (with ≥50% motile cells) was observed in the males treated with the highest dose of aarFsh. In conclusion, these specific recombinant gonadotropins have demonstrated their capacity to induce spermatogenesis and spermiation in vivo in a teleost fish, the European eel.

Feed restriction regime in a rabbit line selected for growth rate alters oocyte maturation manifested by alteration in MSY2 gene expression.

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.

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.

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.

Relationship between sperm quality parameters and the fatty acid composition of the muscle, liver and testis of European eel.

This study looks at the correlations that fatty acids have with different tissues in the European eel (Anguilla anguilla L.) during hormonally-induced sexual maturation, with different sperm quality parameters. In order to evaluate the different dynamics of the use of fatty acids, a categorization of the results from each sperm quality parameter (volume, concentration, motility and velocity) was performed. Low and moderate correlations were observed between muscle tissue and some sperm quality parameters but no high correlations were found. Eicosapentaenoic acid (20:5n3, EPA) in the liver seems to have a role in determining the volume of sperm produced. This can be explained by the fact that EPA is a major requirement in the early phases of sperm production (probably as a component of the spermatozoal membrane). In addition, the levels of α-linolenic acid (18:3-n3, ALA) and linoleic acid (18:2-n6, LA) in the liver decreased when sperm motility increased. In all the tissues, a negative correlation was observed between arachidonic acid (20:4n-6, ARA) and the different sperm velocity parameters. The fact that an increase in the consumption of ARA coincides with an increase in the speed of spermatozoa, highlights the important role that this fatty acid plays not only in sperm production, but also in sperm velocity. All this information could prove useful in the development of suitable broodstock diets to improve sperm quality and subsequently, the larval development of this species.

Effect of exposure to heatwave during blastocyst formation on reproductive performance of female rabbits.

We examined the effect of female exposure to heatwave during blastocyst formation on their reproductive performance and its effect on transcriptome in blastocyst and endometrial tissue. In this study, a total of 72 rabbit does were artificially inseminated and divided into two environmental groups 2 days later: does under conventional conditions (maintained between 14-22°C, n = 29) and does heat stressed in a climatic chamber (maintained between 32-37°C, n = 43). The heat-stressed group were kept under these conditions for 3 days and returned to conventional conditions thereafter. Five days post-insemination, 48 does were slaughtered to collect blastocyst and endometrium samples. mRNA transcripts from OCT-4, VEGF, erbB3, Ifn-É£, HSP70 and HSP90 were analysed by qRT-PCR. At day 12 of gestation, 24 females were examined by laparoscopy to evaluate implanted embryos and at birth the total kits born and individual weights were recorded. Results revealed no gene expression changes in blastocyst and endometrial tissue under heatwave exposure. Moreover, our results demonstrated that rabbit embryos developed from 8-16 cells to blastocyst during a heatwave did not affect implantation rates, total number of kits born and foetal losses. In summary, these results demonstrate that heatwave period is not a critical point in the reproductive performance of rabbits during blastocyst formation.

Does vitrification alter the methylation pattern of OCT4 promoter in rabbit late blastocyst?

Vitrification is replacing slow freezing as the most popular method for oocyte and embryo cryopreservation. However, very little information is available on alterations in epigenetic regulation. Previous studies reported post-implantation effects of vitrification on fetal development and gene expression. This study was conducted to determine if vitrification procedure induce alterations in OCT4 promoter methylation profile which could determine the set point of fetal losses and transcriptomic alterations observed after implantation. Rabbit morulae were recovered at Day 3 of development and vitrified and transferred, or directly transfer, to recipient till Day 6. A conserved regulation region of OCT4 promoter was examined in control and vitrified embryos by bisulfite sequencing and quantitative PCR was used to measure the gene expression. No significant differences were observed in methylation levels or gene expression of OCT4. This work was the first approach in rabbit to the study of possible epigenetic alterations associated with vitrification procedure.

The regulation of aromatase and androgen receptor expression during gonad development in male and female European eel.

This research investigated the regulation of aromatase and androgen receptor gene expression in the brain-pituitary-gonad (BPG) axis of male and female European eels (Anguilla anguilla) during induced sexual maturation. Complete A. anguilla aromatase (aa-cyp19a1) and partial androgen receptor α and ß (aa-ara and aa-arb) sequences were isolated, and qPCR assays were validated and used for quantification of transcript levels for these three genes. Expression levels of the genes varied with sex, tissue and stage of maturation. aa-arb was expressed at higher levels than aa-ara in the pituitary and gonad in both sexes, suggesting aa-arb is the physiologically most important androgen receptor in these tissues. In the female brain, a decrease in aa-ara and an increase in aa-cyp19a1 were observed at the vitellogenic stage. In contrast, a progressive increase in all three genes was observed in the pituitary and ovaries throughout gonadal development, with aa-arb and aa-cyp19a1 reaching significantly higher levels at the vitellogenic stage. In the male pituitary, a decrease in aa-arb and an increase in aa-cyp19a1 were observed at the beginning of spermatogenesis, and thereafter remained low and high, respectively. In the testis, the transcript levels of androgen receptors and aa-cyp19a1 were higher during the early stages of spermatogenesis and decreased thereafter. These sex-dependent differences in the regulation of the expression of aa-ara, aa-arb and cyp19a1 are discussed in relation to the role of androgens and their potential aromatization in the European eel during gonadal maturation.

Vitrification alters rabbit foetal placenta at transcriptomic and proteomic level.

Although numerous studies have demonstrated that cryopreservation alters gene expression, less is known about those embryos that implanted successfully and continued in gestation. To raise the question of the neutrality of this technique, we examine the effects of vitrification through gestation in rabbit before and after the implantation. We monitored the distribution of losses of 569 vitrified morulae, observing that embryos which reach the last pre-implantatory stage are able to implant. However, we found that not all implanted embryos had the ability to continue with their gestation. The results reveal that vitrification decreased foetus and maternal placenta weights at mid-gestation, but led to a higher offspring birth weight. A novel finding is that while no differences in gene expression were detected in pre-implantatory embryos at day 6, vitrification affects a gene and protein expression in the placenta at day 14. Our results for first time reveal strong evidence of modifications in implanted embryos subjected to vitrification, suggesting that the crucial step that vitrified embryos must overcome is the placenta formation. On the basis of these findings, our work leaves the question open as to whether the effects we observed that cause vitrification during foetal development could give rise to some type of physiological or metabolic alteration in adulthood.

Rabbit morula vitrification reduces early foetal growth and increases losses throughout gestation.

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

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 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.

Up-regulation of insulin-like growth factor I and uteroglobin in in vivo-developed parthenogenetic embryos.

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.

Maternal exposure to high temperatures disrupts OCT4 mRNA expression of rabbit pre-implantation embryos and endometrial tissue.

We examined the effect of prolonged high heat stress on reproductive performance and its relationship with gene expression in pre-implantation embryos and endometrial tissue. In experiment 1, primiparous rabbit does were divided into two environments: control does (maintained between 14 and 22°C) and heat-treated does housed in a climatic chamber (maintained between 25 and 35°C). Females were reproducing, and the litter size and live born kits were assessed at 2nd and 3rd partum. In heat-treated does, lower litter size (9.7 ± 0.48 and 11.4 ± 0.50) and fewer live born kits (7.2 ± 0.55 and 10.2 ± 0.57) were observed, although similar ovulation rates and numbers of pre-implantation embryos were noted. In experiment 2, after 3rd partum multiparous non-lactating does from each experimental group were used to obtain pre-implantation embryos and endometrial tissue. mRNA transcripts from OCT-4, VEGF, erbB3, Ifn-É£, HSP70 and HSP90 were analysed by real-time qPCR. Higher values of OCT-4 expression were observed in embryos and endometrial tissue in females reproduced under heat conditions. Moreover, elevated temperatures have been shown to up-regulate VEGF in embryos and down-regulate Ifn-É£ in endometrial tissue. The findings suggest a deleterious temperature effect on litter size and live born kits as a consequence of variation in gene expression pattern of the pre-implantational embryo and the endometrium associated with proliferation and differentiation and probably with implantation and uterine and foetal development during gestation.

Variations in the gene expression of zona pellucida proteins, zpb and zpc, in female European eel (Anguilla anguilla) during induced sexual maturation.

Vertebrate eggs are surrounded by an extracellular glycoprotein coat termed zona pellucida (ZP). Integrity of ZP is critical for a correct embryo development. Two zona pellucida protein genes (zpb and zpc) from European eel were characterized, specific qPCR assays developed and their expression in immature males and females carried out. An experimental group of silver-stage eel females was maintained at 18 °C and hormonally induced to sexual maturation by weekly injections of carp pituitary extract during 12 weeks. Changes in zpb and zpc expression during sexual maturation were studied in liver and ovary by qPCR. In liver, no changes were recorded during hormonal treatment, while in ovary expression of both genes decreased during sexual development. These results are a first step in the characterization of ZP in European eel and in the understanding of the mechanism underlying egg envelope formation.

Comparison of two techniques for the morphometry study on gilthead seabream (Sparus aurata) spermatozoa and evaluation of changes induced by cryopreservation.

The development of powerful software has made possible spermatozoa morphology studies. However, some problems have emerged in relation to protocol standardization to compare results from different laboratories. This study was carried out to compare two techniques commonly used (staining vs phase contrast technique) for the morphometry study of gilthead sea bream spermatozoa using an integrated sperm analysis system (ISAS). Spermatozoa morphometry values were significantly affected by the technique used, and phase contrast technique was found to be the more accurate method, showing lower coefficients of variation on spermatozoa morphometry parameters measurements. Moreover, it has been shown that cryopreservation process produces damage in gilthead sea bream spermatozoa, causing negative effects in sperm parameters as spermatozoa morphometry (a decrease in cell volume), motility (from 95 to 68% motile cells) and viability (from 95 to 87% of live cells), being the addition of freezing medium containing cryoprotectant (DMSO) an important factor that caused the morphometry changes.

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.

mRNA expression in rabbit blastocyst and endometrial tissue of candidate gene involved in gestational losses.

Gestation is a complex process that involves different growth factors, cytokines and adhesion proteins related with embryo development, cellular differentiation and proliferation, embryo-endometrium interaction, angiogenesis, maternal-embryonic recognition and growth development of placenta and embryos. In this study, we examine pre-implantational (at 6 days of gestation) and gestational (at 12 days and total from ovulation to birth) losses in two rabbit lines selected by different criteria (post-weaning daily gain and litter size) and the pattern of a set of candidate transcripts, at 6 days of gestation, related with embryo development and implantation process, such as Oct-4, epidermal growth factor receptor 3 (erbB3), Transforming Growth Factor ß2, Vascular Endothelial Growth Factor and Interferon γ and related with insulin-like growth factors signalling as insulin growth factors I and II and their receptors in rabbit blastocysts and endometrial tissue. Similar pre-implantational losses were obtained in both lines. However, the gestational losses of the line selected by post-weaning daily gain clearly mirrored an increase in losses by 50% at 12 days and at birth (22.4 vs 9.5 and 50.2 vs 25.4, respectively, between line selected by post-weaning daily gain and line selected by litter size). In blastocysts and endometrial tissue at 6 days of gestation qRT-PCR assays indicated that the mean insulin-like growth factor (IGF)-IIR mRNA expression was down-regulated in line selected by post-weaning daily gain. Dysregulation of the IGF-IIR could be potential reasons for induced gestational losses. We conclude that IGF-IIR gene expression in blastocyst and endometrial tissue at 6th day of gestation tends to decline in line selected by post-weaning daily gain. The functional significance related with gestational losses is uncertain.

Parthenogenic blastocysts cultured under in vivo conditions exhibit proliferation and differentiation expression genes similar to those of normal embryos.

Parthenote embryos offer multiple possibilities in biotechnological investigation, such as stem cell research. However, there is still a dearth of knowledge of this kind of embryo. In this study, development and ploidy were analysed in parthenotes under in vitro and in vivo culture conditions. Subsequently, using real-time PCR, the expressions of factor OCT-4, Vascular Endothelial Growth Factor, Epidermal Growth Factor Receptor 3 and Transforming Growth Factor ß2 genes were analysed to compare the embryo types at the blastocyst stage. Development and implantation of parthenote embryos were described after transfer at day 10 of pregnancy. Parthenotes showed similar blastocyst development for both culture conditions and most of the parthenotes produced were diploid. However, parthenotes developed under in vivo conditions showed similar mRNA expression of OCT-4, VEGF and TGF-ß2 to 5 and 6 day old blastocysts. In contrast, parthenotes developed under in vitro conditions had altered the expression pattern of these genes, except for erbB3 mRNA. Finally, transferred parthenotes had the ability to implant but showed severe growth retardation and lesser size. This is the first demonstration of the influence of culture conditions on parthenote mRNA expression. Our study highlights the importance of culture conditions in subsequent uses of parthenotes, such as the production of stem cell lines.

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 fshß and lhß expression, plasma 17ß-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 fshß, lhß, 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 fshß expression and E2 profile in vitellogenic hormonally matured eel females that were more similar to the profile observed in other naturally maturing fish.