Embryonic sex induces differential expression of proteins in bovine uterine fluid.

The bovine endometrium recognizes early embryos and reacts differently depending on the developmental potential of the embryo. However, it is unknown whether the endometrium can distinguish embryonic sex. Our objective was to analyze sexual dimorphism in the uterus in response to male and female embryos. Differentially expressed (DE) proteins, different levels of hexoses, and other embryotrophic differences were analyzed in uterine fluid (UF). Proteomic analysis of day-8 UF recovered from heifers after the transfer of day-5 male or female embryos identified 23 DE proteins. Regulated proteasome/immunoproteasome protein subunits indicated differences in antigen processing between UF carrying male embryos (male-UF) or female embryos (female-UF). Several enzymes involved in glycolysis/gluconeogenesis and antioxidative/antistress responses were up-regulated in female-UF. Fructose concentration was increased in female-UF versus male-UF, while glucose levels were similar. In vitro cultures with molecules isolated from male-UF were found to improve male embryo development compared to female embryos cultured with molecules isolated from female-UF. We postulated that, in vivo, male embryos induce changes in the endometrium to help ensure their survival. In contrast, female embryos do not appear to induce these changes.

Proteome of the early embryo-maternal dialogue in the cattle uterus.

We analyzed embryo-maternal interactions in the bovine uterus on day 8 of development. Proteomic profiles were obtained by two-dimensional difference gel electrophoresis from 8 paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus. Results were contrasted with UF obtained after artificial insemination. We detected 50 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify 38 proteins, obtaining for first time the earliest evidence of involvement of the down-regulated NFkB system in cattle as a pregnancy signature pathway. Embryos enhanced the embryotrophic ability of UF and decreased uterine protein, while blood progesterone was unaltered. Twinfilin, hepatoma-derived growth factor, and synaptotagmin-binding cytoplasmic RNA interacting protein have not previously been identified in the mammalian uterus. TNFα and IL-1B were localized to embryos by immunocytochemistry, and other proteins were validated by Western blot in UF. Glycosylated-TNFα, IL-1B, insulin, lactotransferrin, nonphosphorylated-peroxiredoxin, albumin, purine nucleoside phosphorylase, HSPA5, and NFkB were down-regulated, while phosphorylated-peroxiredoxin, annexin A4, and nonglycosylated-TNFα were up-regulated. The embryonic signaling agents involved could be TNFα and IL-1B, either alone or in a collective dialogue with other proteins. Such molecules might explain the immune privilege during early bovine development.

Efficient derivation of bovine embryonic stem cells needs more than active core pluripotency factors.

Pluripotency can be captured in vitro, providing that the culture environment meets the requirements that avoid differentiation while stimulating self-renewal. From studies in the mouse embryo, two kinds of pluripotent stem cells have been obtained from the early and late epiblast, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), representing the naive and primed states, respectively. All attempts to derive convincing ESCs in ungulates have been unsuccessful, although all attempts were based on the assumption that the conditions used to derive mouse ESCs or human ESC could be applied in other species. Pluripotent cells derived in primates, rabbit, and pig strongly indicate that the state of pluripotency of these cells is, in fact, closer to EpiSCs than to ESCs, and thus depend on fibroblast growth factor (FGF) and Activin signaling pathways. Based on this observation, we have tried to derive EpiSC from the epiblast of bovine elongated embryos as well as ESCs from Day-8 blastocysts. We here show that the core transcription factors Oct4/Sox2/Nanog can be used as markers of pluripotency in the bovine since their expression was restricted to the developing epiblast after Day 8, and disappeared following differentiation of both the ESC-like and EpiSC-like cultures. Although FGF and Activin pathways are indeed present and active in the bovine, it is not sufficient/enough to maintain a long-term pluripotency ex vivo, as was reported for mouse and pig EpiSCs.

Comparison between the traditional Manta net and an innovative device for microplastic sampling in surface marine waters.

Manta nets are commonly used for microplastics sampling although a number of limitations have emerged. In this study we compare the manta net to an innovative microplastic sampler, referred to as MuMi, registered as utility model. The results highlight the large variability that can exist in the outcomes of the different studies due to the lack of harmonization between methods and the differing factors such as sampling mesh size, representativeness or reproducibility of the sampling volumes. Control over the filtered volume is an issue to be improved in trawl sampling methods, while in the MuMi sampler the control over the sampling depth could be improved. Still, MuMi represents a highly advantageous sampling system in terms of ease of operation, lower cost, smaller microplastics target size and greater precision, all while maintaining the representativeness of the collected samples.

Fire hazard after prescribed burning in a gorse shrubland: implications for fuel management.

Prescribed burning is commonly used to prevent accumulation of biomass in fire-prone shrubland in NW Spain. However, there is a lack of knowledge about the efficacy of the technique in reducing fire hazard in these ecosystems. Fire hazard in burned shrubland areas will depend on the initial capacity of woody vegetation to recover and on the fine ground fuels existing after fire. To explore the effect that time since burning has on fire hazard, experimental tests were performed with two fuel complexes (fine ground fuels and regenerated shrubs) resulting from previous prescribed burnings conducted in a gorse shrubland (Ulex europaeus L.) one, three and five years earlier. A point-ignition source was used in burning experiments to assess ignition and initial propagation success separately for each fuel complex. The effect of wind speed was also studied for shrub fuels, and several flammability parameters were measured. Results showed that both ignition and initial propagation success of fine ground fuels mainly depended on fuel depth and were independent of time since burning, although flammability parameters indicated higher fire hazard three years after burning. In contrast, time since burning increased ignition and initial propagation success of regenerated shrub fuels, as well as the flammability parameters assessed, but wind speed had no significant effect. The combination of results of fire hazard for fine ground fuels and regenerated shrubs according to the variation in relative coverage of each fuel type after prescribed burning enabled an assessment of integrated fire hazard in treated areas. The present results suggest that prescribed burning is a very effective technique to reduce fire hazard in the study area, but that fire hazard will be significantly increased by the third year after burning. These results are valuable for fire prevention and fuel management planning in gorse shrubland areas.

Culture system and long-term storage of culture media in the in vitro production of bovine embryos.

The optimum culture system for in vitro matured and fertilised oocytes still remains to be clarified. Culture media (CM) for mammalian embryos are routinely prepared fresh for use and preserved under refrigeration during one or two weeks. The purposes of this work were (1) to compare the efficiency of a synthetic oviduct fluid (SOF) with two different bovine serum albumin (BSA) concentrations (3 and 8 g/L) for the in vitro production of bovine blastocysts, (2) to test the effect of timing on adding fetal calf serum (FCS) to the SOF, and (3) to evaluate the effects on bovine embryo development of freezing and lyophilisation as procedures for preserving the SOF. Supplementation of SOF with 3 g/L BSA increased Day-7 blastocyst expansion rates (18.3 ± 1.6 vs. 14.4 ± 0.7; P < 0.05), although no differences in hatching rates were found. Addition of FCS to SOFaa (SOF with amino acids) medium supplemented with sodium citrate (SOFaaci) at 48 and at 72 h post-insemination (PI) allowed obtaining higher Day-6 embryo development rates than when FCS was added at 18 or 96 h PI (Day-6 morulae + blastocyst rate: 30.0 ± 1.1, 40.8 ± 1.1, 43.9 ± 2.3 and 39.3 ± 0.5 for FCS addition at 18, 48, 72 and 96 h, respectively). Hatching rates were significantly improved when serum was added at 72 h PI. Finally, both refrigeration and lyophilisation appeared as useful cryopreservation procedures for SOFaaci, although a significant loss of its ability to support embryo development, compared to the control fresh culture medium, was observed.

[Effects derived from occupational exposure to hazardous substances in pregnant working women: systematic review]. / Efectos derivados de la exposición laboral en las mujeres trabajadoras embarazadas expuestas a sustancias peligrosas: revisión sistemática.

OBJECTIVE: To review the effects derived from occupational exposure on pregnant working women exposed to hazardous substances. METHODS: Critical analysis of the works retrieved by systematic review in MEDLINE (PubMed), EMBASE, Cochrane Library, Scopus, Web of Science, LILACS and MEDES, up to January 2021. The Search Equation was developed by means of the descriptors «Pregnancy¼, «Hazardous Substances¼ and «Occupational Exposure¼, as well as using related Entry Terms and the filters «Humans¼ and «Adult: 19+ years¼. The quality of the articles was evaluated via the STROBE questionnaire and the level of evidence and the grade of recommendation via the SIGN criteria. RESULTS: Out of the 420 references retrieved (366 digitally and 54 manually) and after applying the inclusion and exclusion criteria, 35 articles were selected. The STROBE evaluation obtained a median of 15.32, and the SIGN criteria, a 2+ level of evidence and a C grade of recommendation. Article obsolescence was high (Burton-Kebler half-life: 22.0; Price index: 0.00%). In 25 out of 35 articles, the exposure is to organic solvents. In 22 articles, congenital anomalies were notified. CONCLUSIONS: The reviewed articles exhibited high obsolescence and a degree of evidence and recommendation that did not allow to fully ensure the validity and reliability of the observations made. The results manifested the association between the exposure to dangerous substances and the occurrence of different damages regarding the health of a pregnant worker and her offspring. The most common exposure was to organic solvents and the most observed effect was congenital alterations.

Objetivo: Revisar los efectos derivados de la exposición laboral en las mujeres trabajadoras embarazadas expuestas a sustancias peligrosas. Métodos: Análisis crítico de los trabajos recuperados mediante revisión sistemática en MEDLINE (PubMed), EMBASE, Cochrane Library, Scopus, Web of Science, LILACS y MEDES, hasta enero 2021. La ecuación de búsqueda se formuló́ mediante los descriptores «Pregnancy¼, «Hazardous Substances¼ y «Occupational Exposure¼, utilizando también los Entry Terms relacionados y los filtros: «Humans¼ y «Adult: 19+ years¼. La calidad de los artículos se evaluó mediante el cuestionario STROBE y el nivel de evidencia y grado de recomendación mediante los criterios SIGN. Resultados: De las 420 referencias recuperadas (366 de forma digital y 54 manual), aplicando los criterios de inclusión y exclusión, se seleccionaron 35 artículos. La evaluación mediante STROBE dio mediana de 15,32 y según los criterios SIGN se obtuvo grado de evidencia 2+ y recomendación C. La obsolescencia fue elevada (semiperiodo de Burton-Kebler: 22,00; índice de Price: 0,00%). En 25 de los 35 estudios revisados la exposición fue a disolventes orgánicos. En 22 artículos se notificó alteraciones congénitas. Conclusiones: Los trabajos revisados presentaron un alto índice de obsolescencia y un grado de evidencia y recomendación que no permitió asegurar por completo la validez y fiabilidad de las observaciones realizadas. Los resultados mostraron la asociación entre la exposición a sustancias peligrosas con la aparición de diferentes daños para la salud de la trabajadora embarazada y su descendencia. La mayor exposición fue a los disolventes orgánicos y el efecto más observado las alteraciones congénitas.

Biological differences between in vitro produced bovine embryos and parthenotes.

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes and in vitro produced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage. In vitro matured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-related POU5F1 and the methylation DNMT3A genes were downregulated in parthenotes. Among pregnancy recognition genes, TP-1 was upregulated in parthenotes, while PGRMC1 and PLAC8 did not change. Expression of p66(shc) and BAX/BCL2 ratio were higher, and p53 lower, in parthenotes. Among metabolism genes, SLC2A1 was downregulated, while AKR1B1, PTGS2, H6PD, and TXN were upregulated in parthenotes, and SLC2A5 did not differ. Among genes involved in compaction/blastulation, GJA1 was downregulated in parthenotes, but no differences were detected within ATP1A1 and CDH1. Within parthenotes, the expression levels of SLC2A1, TP-1, and H6PD, and possibly AKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, through p66(shc) and p53 respectively, and in their mechanisms to control pluripotency and de novo methylation.

Sequential intrapleural administration of fibrinolytic drugs and dornase alfa for empyema management. Treatment protocol based on its physicochemical stability.

OBJECTIVE: Intrapleural administration of fibrinolytics and dornase alfa has been shown in randomized studies to be able to reduce both the  need for surgical debridement of empyema and the average  hospital stay. However, its application in clinical practice is limited,  probably due to the lack of protocols that simplify its administration. The  present study aims to analyze the physicochemical stability of the  simultaneous urokinase and dornase alfa administration for the  subsequent development of a clinical practice use protocol. METHOD: In vitro stability test of urokinase, dornase alfa and a  combination of both. Its stability was evaluated as (i) absence of  particles, (ii) color variation and (iii) pH changes at times 0, 30 minutes,  1, 2 and 4 hours at 37 °C. Each sample was prepared and  analyzed in triplicate. RESULTS: Individual solutions of urokinase and dornase alfa showed slight changes in pH, finding no changes in either color or  presence of suspended particles. The urokinase and dornase alfa  combination was not stable after 2 hours, when turbidity emerged due  to flocculation and phase separation. After 4 hours, precipitate  formation was found. A pro tocol for clinical use was developed based on  urokinase and dornase alfa sequential administration, since it was  not possible to guarantee the physicochemical stability of the simultaneous administration of both drugs. CONCLUSIONS: The physicochemical stability data obtained does not allow to ensure a simultaneous administration of both drugs in a  safe and effective way, thus a sequential administration protocol is  proposed.

Objetivo: La administración intrapleural de fibrinolíticos y dornasa alfa ha demostrado en estudios aleatorizados ser capaz de disminuir la  necesidad de desbridamiento quirúrgico del empiema y los días de  estancia media hospitalaria. Sin embargo, su aplicación en práctica  clínica es limitada, probablemente debido a la falta de protocolos que  simplifiquen su administración. El presente estudio tiene como objetivo  analizar la estabilidad fisicoquímica de la administración simultánea de  uroquinasa y dornasa alfa para el posterior desarrollo de un protocolo de  uso en práctica clínica.Método: Ensayo de estabilidad in vitro de uroquinasa, dornasa alfa y la  mezcla de ambos. Se evaluó su estabilidad como (i) ausencia de partículas, (ii) variación de color y (iii) cambios de pH a tiempos 0,  30 minutos, 1, 2 y 4 horas a 37 °C. Cada muestra se preparó y analizó  por triplicado.Resultados: Las soluciones individuales de uroquinasa y dornasa alfa mostraron cambios ligeros del pH, sin cambios en su color ni  presencia de partículas en suspensión. La mezcla de uroquinasa y  dornasa alfa no fue estable transcurridas 2 horas, mostrando turbidez  por la floculación y sepa ración de fases con formación de precipitado a  las 4 horas. Se desarrolló un protocolo de uso clínico basado en la  administración secuencial de uroquinasa y dornasa alfa, ya que no fue  posible garantizar la estabilidad fisicoquímica de la administración  simultánea de ambos fármacos.Conclusiones: Los datos de estabilidad fisicoquímica no permiten  asegurar la administración simultánea de ambos fármacos de manera  segura y eficaz, por lo que se propone un protocolo de administración  secuencial.

Specific hypertension smartphone application to improve medication adherence in hypertension: a cluster-randomized trial.

Introduction: Digital interventions, such as smartphone applications (apps), are becoming an increasingly common way to support medication adherence and self-management in chronic illness.Aim: To evaluate the effectiveness of the intervention in pharmacological therapeutic adherence in mild to moderate arterial hypertension (AHT), through an app installed on a mobile phone, as well as the degree of control reached by the patient with this tool.Methods: Prospective, randomized controlled trial, full study and multicenter study. Four primary care centers participated. One hundred and fifty-four hypertensive patients under antihypertensive treatment were included. Two groups were established: a control group (CG) with usual intervention (n = 77) and an intervention group (n = 77) (IG), targeting hypertensive people who owned and regularly used a mobile smartphone, specifically using the app called AlerHTA to promote health education and reminder of appointments. There were three visits: initial, 6 and 12 months. Drug adherence was measured by electronic monitors (MEMSs). The primary outcomes were average daily percentage adherence between 80 and 100%, and AHT control.Results: A total of 148 patients finished the study. Mean age was 57.5 ± 9.9. Global adherence was 77.02% (CI = 70.25-83.79) and daily adherence was 74.32% (CI = 67.29-81.35%). Daily adherence was 93.15% and 86.3% in IG, and 70.66% and 62.66% in CG after 6 and 12 months respectively (p < .05). The percentage of uncontrolled patients was 28.3% (CI = 21.05-35.55%). The control of high blood pressure at 12 months was 17.8% and 38.6% for IG and CG respectively (p < .05). The number of patients needed to treat to avoid non-adherence (NNT) was 4.23 patients.Conclusions: The intervention with an app installed on the mobile phones of hypertensive patients favors pharmacological therapeutic adherence and improves the percentage of hypertensive patient control.Trial registration: Spanish Agency of Medicine: EPA-SP UN-HTA-2015-01.

Development and quality of bovine morulae cultured in serum-free medium with specific retinoid receptor agonists.

Retinoids regulate development and differentiation of the bovine blastocyst in vitro, although the underlying mechanisms remain to be clarified. A challenge in reproductive biotechnology is the identification of pathways that regulate early embryonic development and their influence on blastocyst differentiation, apoptosis and survival to cryopreservation as traits of embryo quality. The present paper analyses the effects of short-term exposure (24 h) to retinoids on in vitro-produced bovine morulae. Immature cumulus oocyte complexes were in vitro matured and fertilised. Presumptive zygotes were subsequently cultured in modified synthetic oviduct fluid up to Day 6, in which morulae were randomly allocated to the different experimental groups. The treatments consisted of 0.1 microM LG100268 (LG; a retinoid X receptor agonist), 0.7 microM all-trans retinoic acid (ATRA; a retinoic acid receptor agonist) or no additives. Day 8 blastocyst development was increased in the ATRA-treated group compared with the LG and untreated embryos. In Day 7 embryos, the number of total cells and cells allocated to the trophectoderm were higher in the ATRA-treated group compared with untreated embryos. Apoptosis in the inner cell mass increased after LG treatment, whereas ATRA had no effect. After vitrification and warming, survival and hatching rates of Day 7 blastocysts did not change with retinoid treatment. Within the LG-treated and untreated blastocyst groups, survival and hatching rates were higher for Day 7 than Day 8 embryos; however, Day 8 blastocysts treated with ATRA showed improved hatching rates. In conclusion, treatment of morulae with ATRA in serum-free medium improves embryo development and quality without increasing the incidence of apoptosis and necrosis.

Strategies for improving dabigatran adherence for stroke prevention in patients with non-valvular atrial fibrillation: education and drug intake reminders (FACILITA study).

Objective To assess the efficacy of a mixed intervention, educational, and reminder calendar of the intake, as a strategy to improve therapeutic adherence with dabigatran in patients with non-valvular atrial fibrillation (NVAF). Methods This was a prospective, longitudinal, multi-center study, carried out in 110 specialized healthcare centers in Spain. Seven hundred and twenty-six patients treated with dabigatran prescribed for stroke prevention were included. A cluster randomization was performed based on two groups: (1) Control Group (CG) as usual clinical practice, and (2) Intervention Group (IG) with a mixed strategy: (a) Healthcare education, and (b) Use of a reminder calendar for taking the anticoagulant medication. Three visits took place: baseline and follow-up at 6 and 12 months. Compliance was measured by using electronic monitors (MEMS). Average adherence percentage (%; Average AP) and daily compliance (%; Daily AP) was calculated. A patient was considered adherent when AP was 80-100%. Results Six hundred and twenty-five patients completed the study (315 in the IG and 310 in the CG). Daily AP was 91.97% at 6 months and 91.05% after 12 months in the IG and 82.26% and 82.63% in the CG. Average adherence was 90.79% and 89.20% in the IG and 64.51% and 63.22% in the CG at 6 and 12 months, respectively. Significant differences were observed in the Daily AP and Average AP, with higher percentages in the IG. In the non-adherents group, the number of concomitant drugs, baseline, and 6 months SBP values, 6 and 12 months DBP values, and weight, total cholesterol, and LDL cholesterol were significantly higher. The number needed to treat (NNT) was 3.84 patients to prevent one non-adherence. Conclusions A mixed intervention, consisting of patient education and a simple calendar reminder of drug intake, is an effective strategy to improve dabigatran therapeutic adherence in patients with NVAF. The percentage of adherence with dabigatran was high.

Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability.

The nuclear stage at which oocytes are cryopreserved influences further development ability and cryopreservation affects ultrastructure of both cumulus cells and the oocyte. In this work, we analyze the effects of vitrification at different nuclear and cytoplasmic maturation stages on the oocyte ultrastructure and developmental ability. Culture in TCM199 + PVA with roscovitine 25 M during 24h led to meiotic arrest (MA) in cumulus-oocyte complexes (COCs), while permissive in vitro maturation (IVM) was performed in TCM199, 10% FCS, FSH-LH and 17beta-estradiol for 24 h. Oocytes were vitrified using the open pulled straw method (OPS) with minor modifications. Fresh and vitrified/warmed COCs were fixed as immature, after IVM, after meiotic arrest (MA) and after MA + IVM. Vitrification combined with MA followed by IVM produced the highest rates of degeneration, regardless of the vitrification time. As a consequence, lower proportions of embryos cleaved in these groups, although differences were eliminated at the five-eight cell stage. Development rates up to day 8 were similar in all experimental groups, being significantly lower than those in fresh controls. Only oocytes vitrified after IVM were able to give blastociysts. The morphological alterations observed can be responsible for compromised development. More research is needed to explain the low survival rates of the bovine oocyte after vitrification and warming.

Effects of acetoacetate and D-beta-hydroxybutyrate on bovine in vitro embryo development in serum-free medium.

It is known that the ketone bodies acetoacetate and D-beta-hydroxybutyrate can be metabolized by the early bovine embryo for in vitro development. In the present work, we report experiments leading to the culture of bovine embryos in the absence of serum. In vitro-produced bovine zygotes were cultured in modified synthetic oviduct fluid medium supplemented with acetoacetate derivatives, acetoacetate and D-beta-hydroxybutyrate. Acetoacetate and its derivatives prevented blastocysts from forming in the absence of serum during the whole culture period. However, from Days 6 to 8 of culture in the absence of serum, acetoacetate did not affect development as compared to controls containing lactate and pyruvate or no substrate. Interestingly, D-beta-hydroxybutyrate stimulated blastocyst and expansion development, and allowed lipid mobilization. In feeder cells coculture, embryos produced with D-beta-hydroxybutyrate showed improved hatching. Embryos cultured in D-beta-hydroxybutyrate were viable upon transfer to recipients, although no pregnancies were confirmed later by ultrasonic scanning. The protective effect of serum upon embryos cultured in medium containing acetoacetate is apparently not required in the presence of D-beta-hydroxybutyrate.

Use of two replacements of serum during bovine embryo culture in vitro.

This study evaluated the effect of two commercial serum replacements (Ultroser G and CPSR-3 on in vitro bovine embryo culture. In Experiment 1, zygotes were cultured in SOF+Ultroser G (2, 4 and 6%), SOF+CPSR-3 (2, 4 and 6%), and SOF+5% FCS (control). Blastocyst rates obtained after culturing with Ultroser G were lower than those with FCS. However, blastocyst rates for CPSR-3 were similar to those for serum. In addition, embryos produced in SOF+CPSR-3 had the same proportion inner cell mass number and total cell number as embryos cultured with FCS. In Experiment 2, a combination of serum replacements during different periods showed that treatment before the five-to eight-cell stages had no effect on further embryo development. However, treatments up to the morula stage affected blastocyst formation. The concentration of supplement and the timing of its inclusion in culture markedly affected embryo development. The serum replacement CPSR-3 can supplement embryo culture with blastocyst rates and quality similar to those for serum.

Pregnancy rates and metabolic profiles in cattle treated with propylene glycol prior to embryo transfer.

The objective of this study was to determine the effect of a sustained propylene glycol administration to recipients of frozen/thawed in vivo derived bovine embryos. Heifers were treated with oral propylene glycol for the last 20 days before embryo transfer (n = 142), and untreated as controls (n = 133). Progesterone, insulin, insulin-like growth factor-I, glucose, urea and triglyceride were analysed in blood on Day 0 and Day 7 of the estrous cycle corresponding to embryo transfer. The heifers were selected as recipients when showing progesterone levels <2.0 ng/ml (Day 0) and >2.5 ng/ml (Day 7), according to corpus luteum quality on Day 7 by technicians unaware of animals treated. Within treated animals, significantly more recipients were selected, and increased progesterone, corpus luteum quality, pregnancy and calving rates were recorded. Day 7 progesterone concentrations were higher in heifers treated and transferred. Propylene glycol increased insulin and insulin-like-growth factor-I, but glucose, urea and triglyceride did not vary. Furthermore, insulin-like-growth factor-I, glucose and triglyceride increased at estrous time, but urea decreased and insulin remained unaltered. Together with the sustained gain in pregnancy rates throughout the experiment (2 years), other evidences suggested that the observed effects did not rely on nutritional deficiency. Thus, propylene glycol improved pregnancy rates after embryo-transfer, and progesterone, insulin and insulin-like-growth factor-I are probably involved in this effect.

Metabolomic prediction of pregnancy viability in superovulated cattle embryos and recipients with fourier transform infrared spectroscopy.

We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared spectroscopy (FTIR) metabolomics to identify spectral models predictive of pregnancy outcome. Embryos collected on Day 6 from superovulated cows in 2 countries were individually cultured in synthetic oviduct fluid medium with BSA for 24 h before embryo transfer. Spent CM, blank controls, and plasma samples (Day 0 and Day 7) were evaluated using FTIR. The spectra obtained were analyzed. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the ROC curve (AUC). Endpoints considered were Day 60 pregnancy and birth. High AUC was obtained for Day 60 pregnancy in CM within individual laboratories (France AUC = 0.751 ± 0.039, Spain AUC = 0.718 ± 0.024), while cumulative data decreased the AUC (AUC = 0.604 ± 0.029). Predictions for CM at birth were lower than Day 60 pregnancy. Predictions with plasma at birth improved cumulative over individual results (Day 0: France AUC = 0.690 ± 0.044; Spain AUC < 0.55; cumulative AUC = 0.747 ± 0.032). Plasma generally predicted pregnancy and birth better than CM. These first results show that FTIR metabolomics could allow the identification of embryos and recipients with improved pregnancy viability, which may contribute to increasing the efficiency of selection schemes based on ET.

Ten years after the prestige oil spill: seabird trophic ecology as indicator of long-term effects on the coastal marine ecosystem.

Major oil spills can have long-term impacts since oil pollution does not only result in acute mortality of marine organisms, but also affects productivity levels, predator-prey dynamics, and damages habitats that support marine communities. However, despite the conservation implications of oil accidents, the monitoring and assessment of its lasting impacts still remains a difficult and daunting task. Here, we used European shags to evaluate the overall, lasting effects of the Prestige oil spill (2002) on the affected marine ecosystem. Using δ ¹5N and Hg analysis, we trace temporal changes in feeding ecology potentially related to alterations of the food web due to the spill. Using climatic and oceanic data, we also investigate the influence of North Atlantic Oscillation (NAO) index, the sea surface temperature (SST) and the chlorophyll a (Chl a) on the observed changes. Analysis of δ ¹5N and Hg concentrations revealed that after the Prestige oil spill, shag chicks abruptly switched their trophic level from a diet based on a high percentage of demersal-benthic fish to a higher proportion of pelagic/semi-pelagic species. There was no evidence that Chl a, SST and NAO reflected any particular changes or severity in environmental conditions for any year or season that may explain the sudden change observed in trophic level. Thus, this study highlighted an impact on the marine food web for at least three years. Our results provide the best evidence to date of the long-term consequences of the Prestige oil spill. They also show how, regardless of wider oceanographic variability, lasting impacts on predator-prey dynamics can be assessed using biochemical markers. This is particularly useful if larger scale and longer term monitoring of all trophic levels is unfeasible due to limited funding or high ecosystem complexity.

Seabird feathers as monitors of the levels and persistence of heavy metal pollution after the Prestige oil spill.

We measured heavy metal concentrations in yellow-legged gulls (n = 196) and European shags (n = 189) in order to assess the temporal pattern of contaminant exposure following the Prestige oil spill in November 2002. We analysed Pb, Cu, Zn, Cr, Ni and V levels in chick feathers sampled at four colonies during seven post-spill years (2003-2009), and compared results with pre-spill levels obtained from feathers of juvenile shag corpses (grown in spring/summer 2002). Following the Prestige wreck, Cu (4.3-10 µg g(-1)) and Pb concentrations (1.0-1.4 µg g(-1)) were, respectively, between two and five times higher than pre-spill levels (1.5-3.6 and 0.1-0.4 µg g(-1)), but returned to previous background concentrations after three years. Our study highlights the suitability of chick feathers of seabirds for assessing the impact of oil spills on heavy metal contamination, and provides the best evidence to date on the persistence of oil pollution after the Prestige incident.