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Cardiovascular diseases (CVDs) and diabetes are the primary causes of death in the Philippines. This manuscript reviewed previous studies on the use of predictive analytics for CVD and diabetes risk scoring. This paper also discussed barriers and strategies on how to access/generate available data sets for CVDs and diabetes in the country. CVD and diabetes risk scoring requires the availability of data sets related to such diseases. Although the Philippines has taken strides to implement the Philippine National eHealth solution, such a program does not include strategies toward the use of predictive analytics for CVD and diabetes risk scoring. CVD and diabetes risk scoring research is particularly limited in the Philippines due to challenges related to costs, gaps in policies, and stakeholder involvement. A possible theoretical framework for the analysis and utilization of data sets as well as recommendations and research directions were discussed in this manuscript.

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.

[EN] 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., Funding from the Ministry of Economy, Industry and Competitiveness (Research project: AGL2014-53405-C2-1-P) and Generalitat Valenciana (Research project: Prometeo II 2014/036) is acknowledged. X.G.D. was supported by a research grant from the Ministry of Economy, Industry and Competitiveness (BES-2015-072429). English text version was revised by N. Macowan English Language Service.

[EN] The role of potassium from the seminal plasma and/or the activation media was examined by selectively removing from this media, and by testing the use of 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 pH; in the quiescent stage was also induced. Intracellular pH (pH;) 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 pH; (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., Funded from the SPERMOT project (Spanish Ministry of Science and Innovation, MINECO AGL2010-16009). M.C. Vilchez has a predoctoral grant from UPV PAID Subprogramme 2 (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 (PTA2011-4948-1) and UPV (PTA2011-4948-I). Grants to attend meetings were received from COST Office (AQUAGAMETE COST Action: FA1205).

[EN] The present study was undertaken to evaluate the correlations between the fatty acids in the liver and testis and the plasma levels of the hormonal steroids used during eel spermatogenesis, in order to clarify the physiological roles fatty acids play in the spermatogenetic process. The stages of testis development (S1-S5) were assessed by histological observations in order to classify the different phases of hormonally-induced spermatogenesis and evaluate the possible relationships between the hormones and fatty acids in each stage. The highest plasma levels of 17 beta-estradiol (E2), testosterone (T) and 11-ketotestosterone (11KT) were found in S1, when spermatogonial proliferation occurs. A correlation was found between 17 alpha-20 beta-dihydroxy-4-pregnen-3- one (DHP) levels and some fatty acids during the proliferation and growing phases (S1-2), suggesting that DHP might modulate lipid metabolism in the liver during early spermatogenesis. The DHP levels increased significantly during the growing phase (S2) and remained at high levels throughout the subsequent development stages (S3-S5). Similar to results found in mammals, our results show that in the eel there are regulatory mechanisms, including eicosapentaenoic acid (20:5-n3, EPA) and docosahexaenoic acid (22:6-n3, DHA), which act as modulators in the synthesis of androgens, particularly during the final phase of sperm maturation. Our results suggest that the fact that EPA, ARA and DHA concentrations in the eel testis remain constant/stable during spermiation could be related to the subsequent union of the spermatozoa and the egg. The findings from this research provide new insights for further studies about the possible effect of steroids on desaturase activity and highlight the importance of the effect of lipid metabolism during male eel spermatogenesis. (C) 2014 Elsevier B.V. All rights reserved., Funded by the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no. 245257 (PRO-EEL). D. S. P. had a contract co-financed by MICINN and UPV (PTA2011-4948-I) and received a Shortterm Scientific Mission grant from COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE) to carry out the steroids analyses in Norway.

article i nfo Wild female European eels were matured with CPE (carp pituitary extract) under three thermal regimes, two of which were variable (T10-15 and T15-18, moving from 10 to 15 °C and from 15 to 18 °C, respectively) and one constant, at 18 °C (T18). Before and during hormonal treatment, the eels were sampled and biometric measure- ments were taken. Immunoassays of sex steroids and vitellogenin were performed, as well as qPCR analyses of gene expression (ovarian cyp19a1) and ovarian histology. Prior to the hormonal treatment, the silver eels which had been maintained at 18 °C showed lower 11-KT and E2 plasma levels compared to those maintained at 10 °C. In addition, in the early vitellogenic stage, the androgen and cyp19a1 levels were lower at 18 °C than at 10 °C. Both these results and the positive correlations found between GSI and 11-KT (at the PV stage) and between oocyte diameter and cyp19a1 levels (in the EV stage) suggest that early ovarian development is facili- tated at low temperatures. Vitellogenesis was induced by CPE in all the thermal groups, but progression to the mid-vitellogenic stage was only observed after an accumulation of 900-1200 °D, at 15 or 18 °C, and progression to the late vitellogenic stage was only observed after an accumulation higher than 1300 °D, at 18 °C. Although temperature increased the rate of CPE-induced ovarian development, our results clearly indicate that this in- crease is not linear, but exponential, with acceleration in the increase of GSI at 18 °C from the mid-vitellogenic stage, or after an accumulation of 1300 °D. For the first time, a down-regulation of ovarian cyp19a1 caused by high temperatures in CPE-treated eels was observed. These results demonstrate that temperature can modulate eel ovarian development both before and after exogenous hormonal stimulations, and this knowledge could be used to manipulate the timing of vitellogenesis progression under laboratory conditions.

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. (C) 2013 Elsevier Inc. All rights reserved.

[EN] 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., 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 Generalitat Valenciana through the GV/2007/202 project and ACOMP/2011/229. D.S. Penaranda has a postdoc Grant from the Universitat Politecnica de Valencia (CE-01-10). I.M. and V.G. have predoctoral Grants from Generalitat Valenciana and Spanish MICINN, respectively. F.-A. Weltzien received funding from The Norwegian School of Veterinary Science.

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.

[EN] 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. (C) 2012 Elsevier Inc. All rights reserved., This work was financed by the Spanish Ministry of Science and Innovation (MICINN; AGL2007-64040-C03-00, Project SELECTBREAM). V. Gallego and I. Mazzeo were supported by predoctoral scholarships financed by the Spanish MICINN and Generalitat Valenciana, respectively. D.S. Penaranda had a postdoctoral grant from UPV.

Summary In order to study different storage conditions, dilution ratios and additives for fresh European eel sperm were assayed to determinate the optimum conditions for short-term storage. Two experiments were conducted, analysing the sperm motility and its viability at different times of incubation (1, 4, 7, 11 and 14 days). In the first part of the work, 19 pooled samples were used, and seven in the second experiment. Firstly, exposure to atmospheric and air-limited conditions were compared, registering the highest motility under air-limited conditions, showing no differences to the performance of fresh sperm samples until 96 h of incubation. Also, two different dilutions were checked, with ratios 1 : 10 and 1 : 50, with the latter obtaining better results. A second experiment was performed trying to improve the results obtained in the first experiment. Using air-limited condition and 1 : 50 dilution ratio, the effect of different additives (foetal bovine serum, FBS; N-Tris(hydroxymethyl) methyl-3-aminopropanesulfonic Acid, TAPS; bovine serum albumin, BSA; Glucose) were evaluated. The sperm diluted in the media containing glucose provided motilities without difference in comparison to fresh samples during first week of incubation. At 7 days of storage, the medium containing 300 mm glucose was the only one causing higher motility (77.9 ± 2.5%) in comparison with control medium (50.1 ± 11.5%), demonstrating that the sugar addition was positive to preserve the sperm movement. In conclusion, a new method of eel sperm preservation has been developed, using air-limited conditions, 1 : 50 dilution ratio and glucose as an additive, which provided motilities without significant differences until 7 days of incubation.

Summary Along with sperm quality parameters, the protein profile of European eel seminal plasma was analyzed during induced spermiation (n = 56 samples). Motility, Percentage of live cells, spermatozoa head morphometry and concentration showed low values during the initial weeks of spermiation and maintained high levels throughout the rest of the experiment. The protein profile gradient by SDS-PAGE (4–15%) registered four important electrophoretic bands around 80, 40, 26 and 12 KDa. Three of them showed significant differences in concentration during treatment (80, 40 and 12 KDa), and all of them showed the highest value on the 8th week. Both 80 and 12 KDa bands increased until the 8th week, followed by a progressive decline. One possible explanation for these profiles is that, in the first weeks of treatment, proteins originated from blood plasma are accumulated in the seminal plasma, and from the 8th week some of these proteins are incorporated into the spermatic membranes. The 40 KDa protein band also increased during the first 8 weeks, but maintained high concentrations in the seminal plasma for the rest of the experiment. One result confirms the theory that the presence of proteins in the seminal plasma having a molecular weight lower than 50 KDa increased spermatozoa motility, since the 40 KDa band displayed significantly higher values coinciding with the high percentages of spermatozoa motility. Seminal plasma proteins seem to have an important role in spermatogenesis and spermatozoa movement, but further studies are necessary to discover the identity of these proteins and their precise functions.

Summary As no studies have been carried out to investigate the role of fatty acids in male European eel (Anguilla anguilla) maturation, the main aim of this work was to determine which fatty acids could play a central role in sexual maturation. To this end, European eel males were hormonally induced to sexual maturation and variations in fatty acid composition in muscle, liver, testis and milt were studied. Eels were injected weekly with hCG (Human Chorionic Gonadotropin) for 13 weeks and 10 eels per week were sacrificed. Histological analysis was performed to determine the spermatogenic stage of each male. In muscle, no variations or preferential utilization of fatty acids were detected. In liver, MUFAs (Monounsaturated Fatty Acids) were the most abundant fatty acids in the first weeks, while in the last week PUFAs (Polyunsaturated Fatty Acids) showed the highest concentrations. In testes, PUFAs content increased due to DHA (Docosahexaenoic acid), EPA (Eicosapentaenoic acid) and AA (Arachidonic acid) accumulation, while MUFAs diminished especially due to oleic and palmitoleic acid decrease. Linolenic (EPA and DHA precursor) and linoleic (AA precursor) acids also decreased. As evidenced by histology, main changes in testes matched the appearance of spermatids and first spermatozoa, at the end of meiotic divisions. In milt, no variations were appreciated and PUFAs were the most abundant fatty acids, close to 50%. Due to their increase in testes and the high concentration in milt, PUFAs probably play an important role in the sexual maturation of European eel. Further research is necessary to determine the relationship between feeding, fatty acids and sperm quality.

Summary The aim of this study was to evaluate the effects of different extenders on sea bass (Dicentrarchus labrax) spermatozoa motility and morphology. Six sperm extenders based on inactivator media, DI1 (here named SAN) and Non-Activating Medium (NAM) were tested with European sea bass spermatozoa. The best results were obtained with NAM medium (59.83 mm NaCl, 12.91 mm MgCl2, 1.47 mm KCl, 3.51 mm CaCl2, 20 mm NaHCO3, 0.44 mm glucose) plus 1 and 2% of BSA (NAM1 and NAM2, respectively). The motility of the spermatozoa incubated in those media was similar to the fresh sperm until 48 h (NAM1: 74.3 ± 5.4; NAM2: 78.8 ± 5.8%, and higher than undiluted sperm, 19.1 ± 7.8). We also checked the spermatozoa motility and morphology reactions with some of the best extenders, NAM2 and SAN, and combined them with different concentrations (2, 5, 10%) of three cryoprotectants: methanol, glycerol and dimethyl sulphoxide (DMSO). Glycerol + SAN or NAM2 caused activation of spermatozoa motility, which was lost 5 min later. Methanol and DMSO plus NAM2 extenders resulted in a low activation level and high motility 5 min after incubation, identifying these combinations as good candidates to be used in the cryopreservation of the European sea bass spermatozoa.

Fish sperm quality has traditionally been estimated by subjective evaluation of motility and sperm concentration. Alternative methods for evaluation of sperm quality have been developed in the last decade and enable estimation of spermatozoa head morphometry, membrane integrity and mitochondrial function. Weekly injections of human chorionic gonadotropin (hCG) induced spermiation in farmed male European eels. The milt volume increased from the 5th to 12th weeks. Sperm concentration significantly increased from the 5th week, reaching the highest values at the 8th week, while best motility results were registered at the 9th week of treatment. Coinciding with these intervals, the percentage of dead spermatozoa determined with Hoechst staining showed a reduction in the 8th to 11th weeks of treatment, while the percentage of mitochondrial functionality determined by JC-1 staining did not show a similar pattern. The automatic sperm morphology analysis (ASMA) of the spermatozoa head length, width, area and perimeter showed a significant growth from the 5th to 8th weeks. However, the analysis of isolated descriptive parameters may be difficult to understand because there is a variability in these parameters for each week, making knowledge of the growth kinetic complex. The global size of the spermatozoa head was calculated by applying principal component analysis (PCA), because this method establishes new components that make the interpretation of results easier, allowing a whole interpretation of the changes in the cell morphology. PC1 defines the global head size and shows a significant increase between the 5th and 8th weeks of treatment, showing shorter changes until 12th week. PC2 shows a significant increase in the spermatozoa width from the 5th to 7th weeks. Considering the results of the variations in the principal components defining European eel spermatozoa morphometry, it may be concluded that hCG maturative treatment produced thick cells during the first weeks of spermiation, and subsequent samplings showed an increase in cell width and length. These changes in sperm morphometry coincide with the highest sperm quality assessed as sperm motility and concentration, as well as with the best results obtained in previous studies reporting the best sperm quality between weeks 8 and 10 of hCG treatment. These results support the use of ASMA and Hoechst staining techniques as alternative methods for the evaluation of fish sperm quality.

Five hormonal treatments with human chorionic gonadotropin (hCG) were tested for the induction of maturation and spermiation in male farmed eels. The main aim was to optimize previously used hormonal treatments to achieve shorter induction treatments, longer spermiation periods and/or higher sperm quality. Fish treated for just 3 weeks (treatment E) or until the onset of spermiation (treatment C) showed the worst results, while the treatment consisting of weekly administration of 1.5 IU hCG g−1 fish (treatment A) induced the highest percentage of spermiating males, the highest number of sperm samples and sperm volumes and densities similar to the rest of the treatments (B: half hormone dosage, or D: biweekly administration). Evaluation of the sperm quality was performed by computer-assisted sperm analysis (CASA), considering the percentage of total motile spermatozoa, the percentage of fast and medium-velocity spermatozoa, as well as different motility parameters. Sperm samples from A-D groups showed between 44% and 54% motile spermatozoa, and between 10% and 15% fast spermatozoa, while samples from E-treated males showed 0% motile cells. No significant differences were found in the spermatozoa straight line velocity (VSL), curvilinear velocity (VCL) or the angular velocity (VAP), neither spermatozoa beating cross frequency (BCF) between A–D groups.

The high sperm density, together with the short spermatozoa swimming time, makes European eel sperm manipulation and assessment for quality difficult. Two diluting media (K15 and K30) previously designed for Japanese eel sperm were tested. After 24 h, European eel sperm showed significant reduction in the percentage of motile spermatozoa after activation and different motility parameters (VAP, angular velocity; VCL, curvilinear velocity; VSL, straight line velocity; BCF, beating cross frequency), concluding that these media are not suitable to preserve the sperm of this species. After a hormonal treatment to induce spermiation, sperm volume, density and motility were recorded at weekly samplings. The variation of the osmolality (325–330 mOsm kg−1), pH (8.4–8.6) and the ionic composition (concentration of Na+, K+, Mg2+ and Ca2+) of the seminal plasma were registered. Physio-chemical results were related with sperm quality throughout the treatment, to determine which must be the suitable characteristics of one extender for the sperm of this species, and to find the best conditions to obtain suitable cryopreservation media for European eel sperm. K+ concentration increased, while Ca2+ and Mg2+ concentrations showed a progressive reduction in correlation with the sperm quality improvement. Na+ showed a decreasing, but not significant tendency. P1 and P2 freezing media were designed considering the physio-chemical parameters as well as the ionic composition shown by the best quality sperm samples, and then compared with the previously described solutions, TNK and K30. Sperm quality was determined, checking the percentage of motile spermatozoa and motility parameters using computer-assisted sperm analysis (CASA) software. Samples were frozen after dilution (1:5, 1:20, 1:100) in different freezing media supplemented with 10% dimethyl sulfoxide (DMSO). After thawing, samples frozen with low dilution ratio (1:5) in TNK and P1 media showed higher, although not significant, spermatozoa survival (35.5 ± 14.5 and 36.6 ± 6.7%). The addition of l-α-phosphatidylcholine to the media seems to have a positive effect, as reported in the Japanese eel.

[EN] 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. (C) 2014 Elsevier Inc. All rights reserved., This work was supported by the Valencian Regional Government research programme (Prometeo 2009/125) and the Spanish Research Projects (CICYT AGL2011-29831-C03-01). M.D. Saenz-de-Juano was supported by a research grant from Generalitat Valenciana (Programa VALI+d, ACIF/2011/254).

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‐ɣ, 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‐ɣ 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., This work was supported by the Spanish Research Projects AGL2008–03274 (CICYT). Carmen Naturil was supported by Generalitat Valenciana research programme (Prometeo 2009/125). Estrella Jiménez was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2010/262).

Parthenogenetic embryos are one attractive alternative as a source of embryonic stem cells, although many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. The present work was conducted to investigate the gene expression profile of rabbit parthenote embryos cultured under in vivo conditions using microarray analysis. Transcriptomic profiles indicate 2541 differentially expressed genes between parthenotes and normal in vivo fertilised blastocysts, of which 76 genes were upregulated and 16 genes downregulated in in vivo cultured parthenote blastocyst, using 3 fold-changes as a cut-off. While differentially upregulated expressed genes are related to transport and protein metabolic process, downregulated expressed genes are related to DNA and RNA binding. Using microarray data, 6 imprinted genes were identified as conserved among rabbits, humans and mice: GRB10, ATP10A, ZNF215, NDN, IMPACT and SFMBT2. We also found that 26 putative genes have at least one member of that gene family imprinted in other species. These data strengthen the view that a large fraction of genes is differentially expressed between parthenogenetic and normal embryos cultured under the same conditions and offer a new approach to the identification of imprinted genes in rabbit. © 2012 Naturil-Alfonso et al., This work was supported by Generalitat Valenciana research programme (Prometeo 2009/125). Carmen Naturil was supported by Generalitat Valenciana research programme (Prometeo 2009/125). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.