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Contents Ewes heterozygous for the Fec X R allele (R+) in the bone morphogenetic protein 15 ( BMP15) gene display increased ovulation rate and prolificacy. Besides this phenotypic advantage, the influence of the Fec X R allele on follicle number and size, oocyte competence and in vitro production ( IVP) remains undefined. With these aims, 8 R+ and 8 wild-type (++) ewes were subjected to 2 laparoscopic ovum pick-up ( LOPU) trials (four sessions per trial; two with and two without FSH) and subsequent IVP and fresh embryo transfer. All follicles >3 mm were punctured (n = 1673). Genotype did not significantly affect the number of punctured follicles per ewe and session (10.4 and 10.2 in R+ and ++ untreated ewes, 17.4 and 14.3 in R+ and ++ FSH-treated ewes, respectively), but follicular diameter of R+ ewes was significantly reduced compared with ++ ewes (−0.2 mm in untreated and −0.8 mm in FSH-treated ewes; p < 0.01). R+ ewes showed higher recovery rate and increased numbers of total and suitable cumulus-oocyte complexes for in vitro maturation (IVM). Similar rates of day 8 blastocysts were observed in R+ (36.1%, 147/407) and ++ (32.6%, 100/307) ewes, but the final output of day 8 blastocysts per ewe and session was higher in R+ ewes (+0.75; p < 0.005), without differences in survival rate at birth of the transferred embryos (40.4%, 21/52 vs 36.4%, 16/44, respectively). In conclusion, a higher number of oocytes proven to be competent for in vitro development and embryo survival after transfer are recovered from R+ ewes, despite the lower mean size of their follicles at puncture. [ABSTRACT FROM AUTHOR]

Contents The objective of this study was to test the suitability of a duplex PCR assay for sex and scrapie resistance genotype determination in fresh embryos. Duplex PCR amplified a repetitive and specific fragment of Y chromosome, used for sex diagnosis, and a PrnP fragment. PrnP codons 134 and 156, and codon 171 were genotyped by restriction fragment length polymorphisms and allele-specific PCR, respectively, after re-amplification of PrnP fragment. The specificity of the method was first assessed by testing 359 blood samples from Rasa Aragonesa sheep breed (161 males and 198 females). No amplification failures and total agreement between genotypic and phenotypic sex were found. In the same way, PrnP genotype determination by duplex PCR assay was in agreement with the PrnP animal's genotype established by sequencing. Finally, 73 samples of 1-10 cells from compact morulae were aspirated through the zona pellucida and genotyped for sex and PrnP. The efficiency was 96% when three or more cells were sampled. These results confirm that the duplex PCR assay reported in this work can be used for rapid sex determination in ovine embryos, with a high efficiency and accuracy (96%) when three or more cells are sampled, allowing sexed fresh embryos of known PrnP genotype to be transferred in multiple ovulation and embryo transfer programmes. [ABSTRACT FROM AUTHOR]

In vitro oocyte maturation can be influenced by oocyte source and maturation media composition. The aim of the present study was to compare the efficiency of a defined in vitro maturation medium (TCM199 supplemented with cysteamine and epidermal growth factor; Cys + EGF) with an undefined medium (TCM199 supplemented with follicle-stimulating hormone and follicular fluid; FSH + FF) for in vitro production (IVP) of ovine embryos, using oocytes obtained by laparoscopic ovum pick-up from FSH-stimulated [n = 11; 158 cumulus-oocyte complexes (COCs)] and non-stimulated (n = 16; 120 COCs) live ewes, as well as abattoir-derived oocytes (170 COCs). The produced blastocysts were vitrified and some of them were transferred to synchronized recipients. The best and the worst final yields of embryo IVP observed in this study were obtained using oocytes from FSH-stimulated ewes matured in FSH + FF (41.3%; 33/80) and in Cys + EGF (19.2%; 15/78) medium, respectively (p < 0.01). No significant differences between both media were attained in the blastocyst development rate or in the final yield of embryo IVP using oocytes from non-stimulated ewes or abattoir-derived oocytes. The overall in vivo survival rate of the transferred vitrified blastocysts was 13.1% (8/61), without significant differences between oocyte sources or maturation media. In conclusion, under the experimental conditions of the present study, TCM199 supplemented with cysteamine and EGF is a convenient defined maturation medium for IVP of embryos from oocytes of live non-stimulated ewes or from oocytes of abattoir-derived ovaries. However, the best final yield of embryo IVP observed in this study was attained when oocytes came from FSH-stimulated donors and TCM199 was supplemented with FSH and follicular fluid. [ABSTRACT FROM AUTHOR]

Contents Objectives of this study were to characterize patterns of follicular development in sheep superovulated with purified follicle stimulating hormone (FSH) (OVAGENTM , ICP, Auckland, New Zealand) and to determine its influence on preovulatory events (onset of the oestrus behaviour and timing of the preovulatory luteinizing hormone surge) and ovarian response (ovulation rate and embryo yield). Number and size of all ≥ 23 mm follicles from the first FSH injection to withdrawal of progestagen sponges was determined by transrectal ultrasonography just prior to every FSH injection in nine Manchega ewes superovulated with eight decreasing doses (ml) (1.5 × 3, 1.25 × 2 and 1 × 3) of OVAGEN injected twice daily from 60 h before to 24 h after the withdrawal of 40 mg fluorogestone acetate sponges. Oestrous detection and jugular blood sampling for LH radioimmunoassay were performed every 3 h from 14 to 53 h after sponge removal and ovulation rate and number of embryos were determined 4 days after progestagen withdrawal. Administration of OVAGEN induced a significant rise (p < 0.0005) in the number of follicles ≥ 4 mm in size because of an increased growth in size of follicles from the first FSH injection to sponge removal, an increase in the number of newly detected follicles from 12 to 36 h of the first FSH dose (p < 0.005) and a decrease in regression rate from 24 h (p < 0.001). The number of follicles 2–3 mm in size at first FSH dose (10.4 ± 1.5) was positively correlated with the number of ≥ 4 mm follicles at 0 h (19.0 ± 2.7, p < 0.01). A higher number of ≥ 4 mm follicles at 0 h was related with an earlier appearance of oestrus (31.5 ± 1.5 h, p = 0.08) and LH surge (45.0 ± 2.3 h, p < 0.005), and a higher ovulation rate (18.2 ± 3.8, p < 0.005). On the other hand, the rate of embryo recovery was decreased in ewes with earlier preovulatory LH peaks (p < 0.005), with a shorter... [ABSTRACT FROM AUTHOR]

Developmental failures occurring shortly after blastocyst hatching from the zona pellucida constitute a major cause of pregnancy losses in both humans and farm ungulates. The developmental events occurring following hatching in ungulates include the proliferation and maturation of extra-embryonic membranes - trophoblast and hypoblast - and the formation of a flat embryonic disc, similar to that found in humans, which initiates gastrulation prior to implantation. Unfortunately, our understanding of these key processes for embryo survival is limited because current culture systems cannot sustain ungulate embryo development beyond hatching. Here, we report a culture system that recapitulates most developmental landmarks of gastrulating ovine embryos: trophoblast maturation, hypoblast migration, embryonic disc formation, disappearance of the Rauber's layer, epiblast polarization and mesoderm differentiation. Our system represents a highly valuable platform for exploring the cell differentiation, proliferation and migration processes governing gastrulation in a flat embryonic disc and for understanding pregnancy failures during the second week of gestation. This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The potential of saffron flowers as a source of polyphenols, and in particular anthocyanins, for the extraction of bioactive compounds and the production of a cyanic colorant was analyzed. A microwave pretreatment, prior to the conventional solid-liquid extraction process, was proposed as a feasible intensification step. The effectiveness of microwave pretreatment was assessed in terms of increased yield and improved quality of the final product. The operational variables studied were the pretreatment temperature (60-120 °C) and the solid-liquid ratio (0.30-0.50 g/mL). It was found that the addition of the microwave pretreatment to the conventional process allowed one to reduce extraction time by up to 12 times and to greatly improve the characteristics of the final product, using microwave energy densities as low as 0.16-0.54 kJ/mL. The extract quality was evaluated in terms of polyphenol richness (25% increase), product composition (80% of the anthocyanins was delphinidin), antioxidant capacity (boosted by the pretreatment) and color (variations in red and blue hue depending on conditions). To conclude, a microwave pretreatment in which the material is heated to a temperature of 65 °C with a solvent ratio of 0.30 g/mL was selected as the optimum to maximize process efficiency and product quality.

Epigenetic mechanisms are thought to be involved in the reduced developmental capacity of early prepubertal ewe oocytes compared to their adult counterparts. In this study, we have analyzed the global DNA methylation pattern and in vitro meiotic and developmental competence of oocytes at the germinal vesicle (GV) stage obtained from adult and 3-month-old donors. All oocytes were aspirated from antral follicles with a diameter ≥3 mm, and DNA methylation on 5-methylcytosine was detected by immunofluorescence using an anti-methyl cytosine antibody. The main global chromatin configuration pattern shown by both prepubertal and adult ovine oocytes corresponded to condensed chromatin localized close to the nuclear envelope (the SNE pattern). Immunofluorescence showed that a global bright nuclear staining of 5-methylcytosine (5-mC) occurred in all germinal vesicle stage oocytes and matched the propidium iodide staining pattern. The total fluorescence intensity values of lamb GVs were not lower than those observed in adult GVs. The meiotic competence and cleavage rates were similar in adult and prepubertal oocytes, however, the developmental competence of embryos to reach blastocysts was higher for adult oocytes than lamb oocytes (p<0.0001). In conclusion, our results indicate that adult-size oocytes derived from 3 to 4 month old prepubertal ewes show similar GV morphology and DNA methylation staining patterns to those obtained from adult animals, despite exhibiting a lower developmental competence., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Phenolics present in grapes have been explored as cosmeceutical principles, due to their antioxidant activity and ability to inhibit enzymes relevant for skin ageing. The winemaking process generates large amounts of waste, and the recovery of bioactive compounds from residues and their further incorporation in cosmetics represents a promising market opportunity for wine producers and may contribute to a sustainable development of the sector. The extracts obtained from grape marc and wine lees, using solid-liquid (SL) extraction with and without microwave (MW) pretreatment of the raw material, were characterized in terms of antioxidant activity through chemical (ORAC/HOSC/HORAC) and cell-based (keratinocytes-HaCaT; fibroblasts-HFF) assays. Furthermore, their inhibitory capacity towards specific enzymes involved in skin ageing (elastase; MMP-1; tyrosinase) was evaluated. The total phenolic and anthocyanin contents were determined by colorimetric assays, and HPLC-DAD-MS/MS was performed to identify the main compounds. The MW pretreatment prior to conventional SL extraction led to overall better outcomes. The red wine lees extracts presented the highest phenolic content (3 to 6-fold higher than grape marc extracts) and exhibited the highest antioxidant capacity, being also the most effective inhibitors of elastase, MMP-1 and tyrosinase. The results support that winemaking waste streams are valuable sources of natural ingredients with the potential for cosmeceutical applications.

This article seeks to be a handy document for the academy and the industry to get quickly up to speed on the current status and prospects of biomass pretreatment for biorefineries. It is divided into two biomass sources: vegetal and animal. Vegetal biomass is the material produced by plants on land or in water (algae), consuming sunlight, CO 2 , water, and soil nutrients. This includes residues or main products from, for example, intensive grass crops, forestry, and industrial and agricultural activities. Animal biomass is the residual biomass generated from the production of food from animals (e.g., manure and whey). This review does not mean to include every technology in the area, but it does evaluate physical pretreatments, microwave-assisted extraction, and water treatments for vegetal biomass. A general review is given for animal biomass based in physical, chemical, and biological pretreatments.

Wine lees are rich in anthocyanins (AC), natural colorants with health promoting properties. The extraction kinetics of AC from different wine lees in conventional solid-liquid extraction were studied for the first time. The influence of parameters such as temperature, solid-liquid ratio (R S-L ) and type of solvent (hydro-alcoholic mixtures) was also studied. Furthermore, microwaves (MW) and ultrasounds (US) were used as pre-treatments (a prior step to the conventional extraction) in order to increase AC yield. Maximum extraction yield (2.78 mg MALVIDIN-EQUIVALENTS /g DRY-LEES ) was achieved after 15 min at 25 °C, with a R S-L of 1/10 (g/mL) and with a 50%vol. ethanol mixture. When MW were used AC extraction yield was doubled (6.20 mg MALVIDIN-EQUIVALENTS /g DRY-LEES ) and the required time to achieve a constant yield was reduced (from 15 min to 90 s). Meanwhile, US only shortened extraction time in less proportion (from 15 to 5 min). Putative identification of main extract compounds was performed by LC/MS-MS., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Surface Plasmon Resonance can be used to activate zinc oxide/copper catalysts in order to perform the carbon dioxide hydrogenation reaction by means of light energy, avoiding high-temperature processes. The synthesis and impregnation methods have been designed to fill glass microreactors with ZnO/Cu nanoparticles supported on transparent silica aerogels to maximize the light absorbed by the catalyst. A LED device surrounding the glass microreactors provided white light to activate the catalyst homogeneously throughout the reactor. Temperature, pressure, amount of catalyst and gases flow were studied as possible variables to enhance the process trying to maximize CO₂ conversion rates, achieving the best results working at high pressures. The use of transparent SiO₂ Aerogels as supports for photocatalytic gas phase reactions even under high-pressure conditions is demonstrated.

Aging wine lees are water-wastes produced during the wine aging inside wood barrels that can be considered as alternative sources of bioactive compounds. Phenolic characterization and antioxidant activity (AA) measurements of wines lees solid-liquid extracts have been undertaken on a dry extract (DE) basis. Solvents with different polarities (water, methanol, ethanol, two hydroalcoholic mixtures and acetone) were used. Total phenolic (TPC) and total flavonoid contents (TFC) were determined. The mixture of 75:25(v/v) EtOH:H 2 O showed the highest values with 254 mg GAE /g DE and 146 mg CATE /g DE respectively. HORAC, HOSC and FRAP were used to determine the AA of the extracts being also highest for the mixture of 75:25(v/v) EtOH:H 2 O (4690 µmol CAE /g DE , 4527 µmol TE /g DE and 2197 µmol TE /g DE , respectively). For ORAC method, methanol extract showed the best value with 2771 µmol TE /g DE . Correlations between TPC, TFC, phenolic compounds and AA were determined. Most relevant compounds contributing to AA were identified using data from mass spectrometry, being mainly anthocyanins., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Barley and yeast β-glucans were selected, together with lecithin, to encapsulate resveratrol by emulsification-evaporation method to develop new and safer antifungal formulations. Different emulsification techniques were used: high-shear, high pressure and high pressure and temperature emulsification. Morphology, crystallinity, encapsulation efficiency and in vitro antifungal activity against Botrytis cinerea of the different formulations were evaluated. No significant differences between each emulsification procedure in particle size (below 90nm) and in encapsulation efficiency (70-100%) were observed; only barley β-glucan emulsions showed lower efficiency due to the formation of a gel that retained most of the active compound. A great influence of the emulsification method and the encapsulating material on the crystallinity of the particles was observed. The highest antifungal activity (up to 53% growth inhibition) was obtained by the formulations with yeast β-glucans, indicating an enhanced absorption of encapsulated resveratrol through the cell wall of the fungus at the presence of (1-3, 1-6)-β-glucans., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Recently, supercritical fluid extraction (SFE) has been indicated to be utilized as part of a biorefinery, rather than as a stand-alone technology, since besides extracting added value compounds selectively it has been shown to have a positive effect on the downstream processing of biomass. To this extent, this work evaluates economically the encouraging experimental results regarding the use of SFE during annatto seeds valorization. Additionally, other features were discussed such as the benefits of enhancing the bioactive compounds concentration through physical processes and of integrating the proposed annatto seeds biorefinery to a hypothetical sugarcane biorefinery, which produces its essential inputs, e.g., CO₂, ethanol, heat and electricity. For this, first, different configurations were modeled and simulated using the commercial simulator Aspen Plus ® to determine the mass and energy balances. Next, each configuration was economically assessed using MATLAB. SFE proved to be decisive to the economic feasibility of the proposed annatto seeds-sugarcane biorefinery concept. SFE pretreatment associated with sequential fine particles separation process enabled higher bixin-rich extract production using low-pressure solvent extraction method employing ethanol, meanwhile tocotrienols-rich extract is obtained as a first product. Nevertheless, the economic evaluation showed that increasing tocotrienols-rich extract production has a more pronounced positive impact on the economic viability of the concept.

Subcritical water was employed to fractionate woody biomass into carbohydrates and lignin. Nine urban trees species (hardwood and softwood) from Spain were studied. The experiments were carried out in a semi-continuous reactor at 250 °C for 64 min. The hemicellulose and cellulose recovery yields were between 30%wt. and 80%wt. while the lignin content in the solid product ranged between 32%wt. and 92%wt. It was observed that an increment of solubilized lignin disfavored the hydrolysis of hemicelluloses. It was determined that the maximum extraction of hemicellulose was achieved at 20 min of solid reaction time while the extraction of celluloses not exhibited a maximum value. The hydrolysis of hemicellulose and cellulose would be governed by the hydrolysis kinetic and the polymers accessibility. In addition, the extraction of hemicellulose was negatively affected by the lignin content in the raw material while cellulose hydrolysis was not affected by this parameter., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Beta-glucans and phenolics were extracted from waxy barley using pressurized aqueous ethanol in a stirred batch reactor at 25bar and 500rpm. The effect of temperature (135-175°C), extraction time (15-55min) and ethanol content (5-20%) was evaluated. Temperature had an opposite effect on the extraction of both compounds. The higher the temperature, the lower the β-glucan extraction yield due to fragmentation, but a significant increase on the phenolic recovery was observed. Long extraction times favored the extraction of β-glucans at low temperatures and phenolics at any temperature. The ethanol content was not statistically significant on the β-glucan extraction, but helped to maintain the molecular weight of the extracted β-glucan. To obtain liquid extracts rich in high molecular weight β-glucans and phenolics, mild conditions of 151°C, 21min and 16% ethanol are needed, leading to 51% β-glucan extraction yield with a molecular weight of 500-600kDa and 5mgGAE/g barley., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

At extremely low reaction times (0.02 s), cellulose was hydrolyzed in supercritical water (T=400 °C and P=25 MPa) to obtain a sugar yield higher than 95 wt%, whereas the 5-hydroxymethylfurfural (5-HMF) yield was lower than 0.01 wt %. If the reaction time was increased to 1 s, the main product was glycolaldehyde (60 wt%). Independently of the reaction time, the yield of 5-HMF was always lower than 0.01 wt%. To evaluate the reaction mechanism of biomass hydrolysis in pressurized water, several parameters (temperature, pressure, reaction time, and reaction medium) were studied for different biomasses (cellulose, glucose, fructose, and wheat bran). It was found that the H(+) and OH(-) ion concentration in the reaction medium as a result of water dissociation is the determining factor in the selectivity. The reaction of glucose isomerization to fructose and the further dehydration to 5-HMF are highly dependent on the ion concentration. By an increase in the pOH/pH value, these reactions were minimized to allow control of 5-HMF production. Under these conditions, the retroaldol condensation pathway was enhanced, instead of the isomerization/dehydration pathway., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cellulose hydrolysis can be performed in supercritical water with a high selectivity of soluble sugars. The process produces high-pressure steam that can be integrated, from an energy point of view, with the whole biomass treating process. This work investigates the integration of biomass hydrolysis reactors with commercial combined heat and power (CHP) schemes, with special attention to reactor outlet streams. The innovation developed in this work allows adequate energy integration possibilities for heating and compression by using high temperature of the flue gases and direct shaft work from the turbine. The integration of biomass hydrolysis with a CHP process allows the selective conversion of biomass into sugars with low heat requirements. Integrating these two processes, the CHP scheme yield is enhanced around 10% by injecting water in the gas turbine. Furthermore, the hydrolysis reactor can be held at 400°C and 23 MPa using only the gas turbine outlet streams., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Hybrid lipid-polymer particles are gaining increasing interest to be applied as drug delivery systems due to their greater stability in biological fluids and enhanced cellular uptake of drugs. However, a major drawback is the fact that these particles are usually produced through techniques that use organic solvents. This work studies the possibility of producing for the first time hybrid particles composed by lipid multicores enveloped in a polymeric layer through Particles from Gas Saturated Solutions (PGSS(®)), without using organic solvents. An oil-in-water emulsion, composed by Gelucire 43/01™ (GEL) as the discontinuous phase and by an aqueous polyethylene glycol 4000 (PEG) solution as the continuous phase, was successfully precipitated by PGSS(®). Operating conditions that ensured the stability of the CO2 saturated emulsion were previously evaluated. The resulting PEG-GEL particles present a spherical-like morphology constituted by several lipid cores encapsulated into a polymeric shell. The applicability of these structured particles to be used as drug delivery system (DDS) was studied by using ketoprofen, a nonsteroidal anti-inflammatory drug (NSAID), as model drug. The particles provided an initial burst release of the drug due to the progressive dissolution of the external layer of PEG, followed by a controlled release of the NSAID from the GEL cores., (Copyright © 2014. Published by Elsevier B.V.)

A novel plasmonic reactor concept is proposed and tested to work as a visible energy harvesting device while allowing reactions to transform CO2 to be carried out. Particularly the reverse water gas shift (RWGS) reaction has been tested as a means to introduce renewable energy into the economy. The development of the new reactor concept involved the synthesis of a new composite capable of plasmonic activation with light, the development of an impregnation method to create a single catalyst reactor entity, and finally the assembly of a reaction system to test the reaction. The composite developed was based on a Cu/ZnO catalyst dispersed into transparent aerogels. This allows efficient light transmission and a high surface area for the catalyst. An effective yet simple impregnation method was developed that allowed introduction of the composites into glass microchannels. The activation of the reaction was made using LEDs that covered all the sides of the reactor allowing a high power delivery. The results of the reaction show a stable process capable of low temperature transformations.

Bioactive compounds of wine making by-products are of interest in food, cosmetics and pharmaceutical industries. Extraction of antioxidants under mild conditions is time-consuming, giving ground to the development of intensification processes where the operation at high temperature may deteriorate extract quality. This study examined thermal degradation of grape marc and its filtered extract (80, 100 and 150°C). The decrease in anthocyanin content was modelled under non-isothermal conditions by first order kinetics, using the Arrhenius equation. Simulated degradation under isothermal heating showed that the grape marc is more sensitive by one order of magnitude to heat than the filtered extract. This tendency was also confirmed by analyses of the total phenolic content and antioxidant activity. It is suggested that an optimal combination of temperature, treatment time and also raw material environment could be found in process intensification., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Embryo biotechnologies contribute significantly to the genetic enhancement of livestock, although their efficiency remains limited in sheep, mainly owing to variable ovarian responses to gonadotropins. At present, anti-Müllerian hormone (AMH), which is produced by the granulosa cells of the small antral follicles, is a reliable endocrine marker of the ovarian follicle reserve in many species. The expression of AMH in granulosa cells was shown to be stimulated by bone morphogenetic proteins (BMPs) in vitro, so a mutation affecting the BMP15 gene might modulate AMH production in vivo. The present study aimed to assess plasma AMH concentrations before puberty in two groups of Rasa Aragonesa ewes that were carrying (R+) or not carrying (++) the prolific FecX(R) allele and to relate them with their AMH concentrations at adulthood. Additionally, we sought to establish in both genotypes whether AMH measurements during a laparoscopic ovum pick-up (LOPU) program could be predictive of the number of ovarian follicles (≥3 mm) and recovered cumulus-oocyte complexes (COCs). No differences in AMH were found between the R+ and ++ ewes before puberty or during the adult age. Before puberty, the AMH concentration tended to increase from 3 to 4.5 months and to decline at 6 months to levels similar to those observed later in adults (333.8 ± 73.3, 483.2 ± 135.5, and 184.1 ± 38.2 pg/mL, respectively; P < 0.1), showing a large variability between individuals and between ages. A relationship between the AMH concentrations before puberty and during adulthood was not found, likely reflecting different follicular growth dynamics. In adults, the AMH concentration at the beginning of the FSH treatment was strongly correlated with the number of punctured follicles at LOPU in R+ and ++ ewes (r = 0.75 and 0.78, respectively; P < 0.001), and it was possible to accurate determine AMH cutoff values for both genotypes to identify high-responding ewes. On average, 5.1 extra follicles and 2.7 extra COCs were expected per each 100 pg/mL increase in AMH (P < 0.0001 and P < 0.01, respectively). The repeatability of AMH concentration from session to session was 0.70 (P < 0.0001). Our results demonstrated that, regardless of age, the presence of the FecX(R) allele did not affect plasma AMH levels. During adulthood, AMH proved to be a good predictor of the ovarian response to FSH stimulation. Such an indicator could therefore be used to improve the performance of embryo biotechnologies in sheep., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ewes heterozygous for the FecX(R) allele (R+) in the bone morphogenetic protein 15 (BMP15) gene display increased ovulation rate and prolificacy. Besides this phenotypic advantage, the influence of the FecX(R) allele on follicle number and size, oocyte competence and in vitro production (IVP) remains undefined. With these aims, 8 R+ and 8 wild-type (++) ewes were subjected to 2 laparoscopic ovum pick-up (LOPU) trials (four sessions per trial; two with and two without FSH) and subsequent IVP and fresh embryo transfer. All follicles >3 mm were punctured (n = 1673). Genotype did not significantly affect the number of punctured follicles per ewe and session (10.4 and 10.2 in R+ and ++ untreated ewes, 17.4 and 14.3 in R+ and ++ FSH-treated ewes, respectively), but follicular diameter of R+ ewes was significantly reduced compared with ++ ewes (-0.2 mm in untreated and -0.8 mm in FSH-treated ewes; p < 0.01). R+ ewes showed higher recovery rate and increased numbers of total and suitable cumulus-oocyte complexes for in vitro maturation (IVM). Similar rates of day 8 blastocysts were observed in R+ (36.1%, 147/407) and ++ (32.6%, 100/307) ewes, but the final output of day 8 blastocysts per ewe and session was higher in R+ ewes (+0.75; p < 0.005), without differences in survival rate at birth of the transferred embryos (40.4%, 21/52 vs 36.4%, 16/44, respectively). In conclusion, a higher number of oocytes proven to be competent for in vitro development and embryo survival after transfer are recovered from R+ ewes, despite the lower mean size of their follicles at puncture., (© 2013 Blackwell Verlag GmbH.)

An increasing interest in biomass as a renewable feedstock for the chemical industry has risen over the last decades, and glucose, the monomer unit of cellulose, has been widely studied as a source material to produce value-added products such as carboxylic acids, mainly gluconic and formic. In this work, the non-catalysed wet oxidation of glucose using hydrogen peroxide has been analysed, obtaining molar yields to gluconic and formic acids up to 15% and 64%, respectively. Glucose conversion was generally between 40 and 50%, reaching over 80% under the highest temperature (200°C). An appropriate choice of temperature can tune product distribution as well as reaction rates. The interaction of the wet oxidation with an electrolytic reaction was also analysed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In this study, microwave treatment is analyzed as a way to accelerate the hydrolysis in anaerobic digestion of municipal wastewater sludge. The influence of the absorbed energy, power and athermal microwave effect on organic matter solubilization and biogas production has been studied. In addition, a novel method that considers the absorbed energy in the microwave system is proposed, in order to obtain comparable experimental results. The absorbed energy is calculated from an energy balance. The highest solubilization was achieved using 0.54 kJ/ml at 1000 W, where an increment of 7.1% was observed in methane production, compared to the untreated sample. Using a higher energy value (0.83 kJ/ml), methane production further increased (to 15.4%), but solubilization decreased. No power influence was found when 0.54 kJ/ml was applied at 1000, 600 and 440 W. Microwave heating was compared to conventional heating in two different experimental setups, providing similar methane yields in all cases., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The hydrothermal flame is a new method of combustion that takes place in supercritical water oxidation reactions when the temperature is higher than the autoignition temperature. In these conditions, waste can be completely mineralized in residence times of milliseconds without the formation of by-products typical of conventional combustion. The object of this work is to study the hydrothermal flame formation in aqueous streams with high concentrations of recalcitrant compounds: an industrial waste with a high concentration of acetic acid and various concentrated solutions of ammonia. A tubular reactor with a residence time of 0.7 s was used. Oxygen was used as the oxidant and isopropyl alcohol (IPA) as co-fuel to reach the operation temperature required. The increase of IPA concentrations in the feeds resulted in a better TOC removal. For mixtures containing acetic acid, 99% elimination of TOC was achieved at temperatures higher than 750 °C. In the case of mixtures containing ammonia, TOC removals reached 99% while maximum total nitrogen removals were never higher than 94%, even for reaction temperatures higher than 710 °C. Ignition was observed at concentrations as high as 6% wt NH(3) with 2% wt IPA while at IPA concentrations below 2% wt IPA, the ammonia did not ignite., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Two experiments have been performed to clone the bucardo, an extinct wild goat. The karyoplasts were thawed fibroblasts derived from skin biopsies, obtained and cryopreserved in 1999 from the last living specimen, a female, which died in 2000. Cytoplasts were mature oocytes collected from the oviducts of superovulated domestic goats. Oocytes were enucleated and coupled to bucardo's fibroblasts by electrofusion. Reconstructed embryos were cultured for 36h or 7d and transferred to either Spanish ibex or hybrid (Spanish ibex malex domestic goat) synchronized recipients. Embryos were placed, according to their developmental stage, into the oviduct or into the uterine horn ipsilateral to an ovulated ovary. Pregnancy was monitored through their plasmatic PAG levels. In Experiment 1, 285 embryos were reconstructed and 30 of them were transferred at the 3- to 6-cells stage to 5 recipients. The remaining embryos were further cultured to day 7, and 24 of them transferred at compact morula/blastocyst stage to 8 recipients. In Experiment 2, 154 reconstructed embryos were transferred to 44 recipients at the 3- to 6-cells stage. Pregnancies were attained in 0/8 and 7/49 of the uterine and oviduct-transferred recipients, respectively. One recipient maintained pregnancy to term, displaying very high PAG levels. One morphologically normal bucardo female was obtained by caesarean section. The newborn died some minutes after birth due to physical defects in lungs. Nuclear DNA confirmed that the clone was genetically identical to the bucardo's donor cells. To our knowledge, this is the first animal born from an extinct subspecies.

Ionic liquids (IL) are receiving increasing attention due to their potential as "green" solvents, especially when used in combination with SC-CO2. In this work liquid-vapor equilibria of binary mixtures of CO2 with two imidazolium-based ionic liquids (IL) with a nitrate anion have been experimentally determined: butylmethylimidazolium nitrate (BMImNO3) and hydroxypropylmethylimidazolium nitrate (HOPMImNO3), using a Cailletet apparatus that operates according to the synthetic method. CO2 concentrations from 5 up to 30 mol % were investigated. It was found that CO2 is substantially less soluble in HOPMImNO3 than in BMImNO3. Since these ILs are very hygroscopic, water easily can be a major contaminant, causing changes in the phase behavior. In case these Ils are to be used in practical applications, for instance, together with CO2 as a medium in supercritical enzymatic reactions, it is very important to have quantitative information on how the water content will affect the phase behavior. This work presents the first systematic study on the influence of water on the solubility of carbon dioxide in hygroscopic ILs. It was observed that the presence of water reduces the absolute solubility of CO2. However, at fixed ratios of CO2/IL, the bubble point pressure remains almost unchanged with increasing water content. In order to explain the experimental results, the densities of aqueous mixtures of both ILs were determined experimentally and the excess molar volumes calculated.

Sex specific sequence variability of the amelogenin gene has been used for sex determination in the family of Bovidae. In our study, suitability and reliability of the amelogenin gene for ovine sex determination in embryos was studied. The specificity of the method was previously demonstrated by testing 579 blood samples from several Spanish sheep breeds (161 males and 198 females). No amplification failures and very high agreement between genotypic and phenotypic sex was found (578/579), in contrast to humans, where errors in sex determination has been reported because of mutations in AMELX or AMELY genes. However, one female animal showed a male genotypic sex, being the most plausible explanation that a recombination event has happened during the meiosis. In our study only 0.17% (1/579) of the samples tested has been misdiagnosed using the amelogenin gene. Finally, 1-10 cells from each of 67 compact morulae were aspirated through the zona pellucida, and genotyped for sex determination. The efficiency in sex determination was 95 and 98% when more than two and more than three cells were sampled, respectively. The total time required for the genetic test, was less than 4h. These results confirm that the amelogenin gene can be used for rapid sex determination in ovine embryos, with a high efficiency and accuracy (100%) when three or more cells are sampled, allowing transferring sexed fresh embryos in MOET programmes. To our knowledge, this was the first time that sex determination using the amelogenin gene was performed in ovine embryos.

Supercritical fluid techniques for materials precipitation have been proposed as an alternative to conventional precipitation processes as they allow to improve the performance of these processes in terms of reduction of particle size and control of morphology and particle size distribution, without degradation or contamination of the product. These techniques have received much attention during the last years, and their feasibility and performance have been experimentally demonstrated for many substances. One of the main pending tasks is the development of a systematic procedure for the design and scale-up of these processes. This requires not only empirical knowledge, but also information about the fundamentals of the process. This work aims to review the published literature dealing with a fundamental investigation or modeling of supercritical fluid precipitation processes.

In the current study we investigated whether the developmental status of the two largest follicles (LF1 and LF2) at the time of administration of the first two doses (0 and 12 h) of FSH of a superovulatory treatment influences periovulatory events and embryo yields in sheep. A larger size of LF1 was negatively correlated with embryo recovery (r=-0.608 for 0 h and r=-523 for 12 h, p<0.05), fertilization (r=-0.464 for 12 h, p<0.05) and viability (r=-0.775 for 12 h, p<0.005). Embryo viability rates were also lower when a higher difference between LF1 and LF2 (r=-0.839 for 0 h and r=-0.761 for 12 h, p<0.01) and a smaller size of LF2 (r=0.877 for 0 h and r=0.622 for 12 h, p<0.01) were observed. This indicates the existence of a limit in the follicular size that will be able to give rise a viable embryo. Conversely, a larger size of LF2 at the time of administration of the first two FSH doses was correlated with reduced recovery rates (r=-0.884 for 0 h and r=-0.706 for 12 h, p<0.01), whilst a decreasing size of LF1 and LF2 was correlated with an increased ovulation rate and recovered embryos. The dominance effect appeared to affect the timing of the preovulatory LH surge. Ewes with a higher difference between LF1 and LF2 displayed earlier LH surges (r=-0.420 for 0 h and r=-0.401 for 12 h, p<0.05) which were related to a higher number of non viable embryos (r=-0.777, p<0.05). The fact that superovulatory yields were affected by, both LF1 and LF2 supports the hypothesis of co-dominance effects in sheep.

The supercritical water oxidation (SCWO) is a technology that takes advantage of the special properties of water in the surroundings of critical point of water to completely oxidize wastes in residence times lower than 1 min. The problems caused by the harsh operational conditions of the SCWO process are being solved by new reactor designs, such as the transpiring wall reactor (TWR). In this work, the operational parameters of a TWR have been studied for the treatment of an industrial wastewater. As a result, the process has been optimized for a feed flow of 16 kg/h with feed inlet temperatures higher than 300 degrees C and transpiring flow relation (R) between 0.2 and 0.6 working with an 8% (w/w) isopropanol (IPA) as a fuel. The experimental data and a mathematical model have been applied for the destruction of an industrial waste containing acetic acid and crotonaldehyde as main compounds. As the model predicted, removal efficiencies higher than 99.9% were obtained, resulting in effluents with 2 ppm total organic carbon (TOC) at feed flow of 16 kg/h, 320 degrees C of feed temperature and R = 0.32. An effluent TOC of 35 ppm under conditions feed flow of 18 kg/h, feed inlet temperatures of 290 degrees C, reaction temperatures of 570 degrees C and R = 0.6.

Embryo production is a useful tool for ex situ conservation of endangered species and breeds, despite a high variability in the ovarian response to superovulatory treatments. The current study evaluated the incidence and mechanisms of genetic factors in such variability, by determining the pharmacokinetics and pharmacodynamics of a standard treatment with ovine FSH (oFSH) in two endangered Spanish sheep breeds (Rubia del Molar, R, and Negra de Colmenar, N) in comparison to Manchega ewes (M, control group). In the first experiment, pharmacokinetics of an i.m. single dose of 1.32 mg of oFSH was evaluated in seven animals of each breed. Plasma FSH concentrations reached their maximum at 4h post-administration in all the ewes, but several of the kinetic parameters (plasma FSH concentration at 4h post-administration, maximum plasma FSH concentration, C(max), and both the area under the plasma concentration-time curve extrapolated to the infinite, AUC(inf), and to the last moment of sampling, AUC(last)) were higher in the N group. In the second trial, 10 animals of each breed were superovulated using eight decreasing doses of oFSH (3 x 1.32 mg, 2 x 1.10 mg, and 3 x 0.88 mg). The R group, when compared to N and M, showed both a higher number of corpora lutea (13.7+/-0.6 versus 10.0+/-0.4 in N and 9.8+/-0.6 in M, P<0.05 for both) and embryos (7.9+/-0.8 versus 4.3+/-0.4 in N, P<0.05, and 6.7+/-0.5 in M, n.s.). Evaluation of pharmacokinetic and dynamic parameters showed that, although there was a trend for a higher hormone availability in R sheep, mean FSH plasma concentrations were similar between breeds (0.54+/-0.08 ng/ml for R, 0.45+/-0.05 ng/ml for N and 0.35+/-0.05 ng/ml for M). However, differences were found in the number of preovulatory follicles growing in response to the FSH treatment between R (24.4+/-2.2), M (18.9+/-1.5, n.s.) and N sheep (14.1+/-1.4; P<0.01). Thus, differences in embryo yields between breeds would be related to differences in the pattern of follicular growth in response to FSH treatment.

This study evaluated the final output and quality of in vitro produced blastocysts derived from in vivo recovered sheep embryos cultured at various early developmental stages to blastocyst. A total of 270 embryos were recovered from the oviduct, at different days of the early luteal phase, and were classified into three different developmental stages: 2- to 4-cell (n = 93); 5- to 8-cell (n = 92) and 9- to 12-cell (n = 85). The effect of culture conditions was studied, at the same time, by randomly allocating the embryos to one of four groups: three groups of culture with fresh oviduct monolayers (2, 4 and 5 days old) and a fourth group with 2-day monolayers derived from frozen-thawed oviduct cells. Two control groups were established: first, embryos cultured in semi-defined medium (n = 29) and, second, blastocysts obtained in vivo and cryopreserved (n = 43). Influence on blastocyst yield of embryo developmental stage at the start of culture was statistically significant (p < 0.001). Two- to four-cell embryos showed a significantly lower developmental rate (67.7%) than the 5- to 8-cell (83.6%; p < 0.001) and 9- to 12-cell groups (90.5%; p < 0.0001) and lower quality in terms of blastocyst cryotolerance (56.0 vs. 83.7%; p < 0.005). There were no detected effects relating to the age or handling of the monolayer on the embryo developmental rate, but the day of blastocyst appearance was different between embryos cultured on monolayers derived from fresh or frozen-thawed cells (p < 0.0001); the main influence was on the group of 9- to 12-cell embryos (p < 0.0001). Current results confirm the temporal sensitivities of sheep embryos to in vitro culture, regardless of the culture conditions.

This study evaluates the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from sheep treated with GnRH antagonists (GnRHa) and high doses of FSH. Eighteen Sarda ewes were treated with progestagen sponges (day 0). On day 7, 10 ewes received 3 mg of GnRHa s.c., while 8 served as control receiving saline. On day 10, all animals were treated with 96 IU of ovine FSH in four equal doses given i.m. every 12 h. We monitored follicular development by ultrasonography, twice daily from day 7 to 11, and found that GnRHa induced a significant increase in the number of total follicles in 72 h (11.7+/-0.9 to 21+/-2.4, r(2)=0.598, P<0.0001), while this number remained stable in control sheep. We found that FSH induced a significant rise in the number of follicles in both groups; but always higher (P<0.05) in GnRHa treated sheep, confirming that GnRHa enhances ovarian response to exogenous FSH stimulation. Twelve hours after the last FSH dose, oocytes were collected by OPU. Recovery percentage, morphological quality, ability to resume meiosis, fertilization and cleavage were similar in oocytes from treated and untreated sheep. However, the final blastocysts output was lower in GnRHa group (10.1% versus 27.4% in control group; P<0.05). In addition, re-expansion rates after vitrification, thawing and in vitro culture were lower in GnRHa treated ewes, although differences did not reach statistical significance (55.5% versus 74.1% in GnRHa treated and in control sheep, respectively).

Efficiency of superovulatory protocols is affected by the occurrence of reproductive abnormalities, such as the presence of anovulatory follicles. The objective of current study was to assess the incidence and possible causes of anovulatory follicles in superovulated sheep, in order to characterize the endocrine functionality of these follicles in terms of estradiol production and to evaluate their relationship with development of embryos from other follicles. The number and size of all follicles present in the ovaries of 12 sheep treated with a superovulatory FSH step-down treatment was assessed by ultrasonography. On Day 3 after subsequent estrus behaviour, the number of corpora lutea and anovulatory follicles were recorded and the fluid of anovulatory follicles >or=5mm in size was aspirated and assayed for estradiol. At once, embryos were recovered to evaluate their viability. In current study, anovulatory structures averaged 34.6% of the follicles developing to preovulatory sizes. The number of anovulatory follicles was determined by the existence of follicular dominance effects, since they increased with a higher difference in size between the largest and the second largest follicle at the beginning of the superovulatory treatment (P<0.05, r(2)=0.420). Most of the anovulatory follicles showed signs of functionality failures, indicated by a low mean estradiol concentration (9.9+/-1.1 ng/ml). However, a 22.4% of them were highly estrogenic (>200 ng/ml) and their permanence beyond the ovulation was related to a drop in the embryo viability rate (P<0.005), leading to decreased final superovulatory yields.

This study evaluated the effect of freezing-thawing procedures on the viability of sheep embryos cryopreserved at various developmental stages. The survival rates of frozen-thawed embryos were compared with non-frozen counterparts. Embryos were recovered from the oviduct and uterus, at different days of the early luteal phase, and were classified at six different developmental stages: 2- to 4-cell (n = 72), 5- to 8-cell (n = 73), 9- to 12-cell (n = 70), early morulae (n = 42), morulae (n = 41), and blastocyst (n = 70). For each early cleavage stage and blastocysts, approximately half of the embryos, were frozen immediately by slow freezing with an ethylene glycol-based solution. The remaining embryos were cultured to the hatched blastocyst stage. All morulae and compact morulae were frozen after recovery with the same protocol. Cryoprotectants were removed using 1M sucrose solution, and then warmed the embryos were cultured to the hatched stage in a standardized in vitro culture. Embryo developmental stage had a significant effect on the ability to hatch following freezing (P<0.0001). The cryotolerance of the embryos fitted a regression (r2 = 0.908), increasing linearly from 2- to 4-cell embryos (17.1%) to morula stage (46.3%) and in a quadratic regression from the morula to the blastocyst stage (83.7%). Frozen early cleavage stage embryos had a significantly lower viability than their fresh counterparts (23.1 vs 83.1%; P<0.0001), with a similar rate of viability between fresh or frozen blastocysts (92.5 vs 83.7%). In conclusion, early sheep embryos are very sensitive to freezing per se and the survival rates following conventional freezing improve as embryo developmental stage progresses.

The objective of this study was to characterize follicular development, onset of oestrus and preovulatory LH surge, and in vivo embryo yields of sheep superovulated after treatment with a single dose of 1.5mg of GnRH antagonist (GnRHa). At first FSH dose, ewes treated with GnRH antagonist (n=12) showed a higher number of gonadotrophin-responsive follicles, 2-3mm, than control ewes (n=9, 13.5+/-3.8 versus 5.3+/-0.3, P<0.05). Administration of FSH increased the number of >or=4mm follicles at sponge removal in both groups (19.3+/-3.8, P<0.0005 for treated ewes and 12.7+/-5.4, P<0.01 for controls). Thereafter, a 25% of the GnRHa-treated sheep did not show oestrous behaviour whilst none control sheep failed (P=0.06). The preovulatory LH surge was detected in an 88.9% of control ewes and 66.7% of GnRHa-treated sheep. A 77.8% of control females showed ovulation with a mean of 9.6+/-0.9 CL and 3.3+/-0.7 viable embryos, while ewes treated with GnRHa and showing an LH surge exhibited a bimodal distribution of response; 50% showed no ovulatory response and 50% superovulated with a mean of 12.2+/-1.1 CL and 7.3+/-1.1 viable embryos. In conclusion, a single dose of GnRHa enhances the number of gonadotrophin-dependent follicles able to grow to preovulatory sizes in response to an FSH supply. However, LH secretion may be altered in some females, which can affect the preovulatory LH surge and/or can weak the terminal maturation of ovulatory follicles.

This report offers the results of two experiments developed to test possible benefitial effects of the presence of corpus luteum (CL) on in vivo and in vitro sheep embryo production; using two different breeds treated with two different protocols by two different teams at two different centres. In the first trial, estrus was synchronized in 11 ewes with two doses of cloprostenol, 10 days apart. On day 1 after estimated ovulation, sheep were treated with progestagen sponges and superovulated with eight decreasing doses (26.4 units NIH-FSH-S1 x 3, 22.0 units x 2, and 17.6 units x 3) of ovine FSH injected twice daily. Ovulation rate and number of embryos obtained in vivo were compared to those from 12 control ewes without cloprostenol treatment. Presence of a CL improves the number of transferable embryos (7.4+/-0.6 versus 4.1+/-0.6 in control ewes, P < 0.05). The second trial investigated the effects of the presence of CL on embryos produced in vitro from six ewes bearing CL and six ewes without CL at start a superovulatory treatment consisting of 96 units of ovine FSH administered in four equal doses given every 12 h. There were not detected effects of the CL on the number and size of follicles or in the number, morphology and ability to resume meiosis of their oocytes. However, oocytes from ewes with CL showed higher rates of fertilization (73.5 versus 45.5%, P < 0.005), higher development to blastocyst (35.8 versus 19.3%, P < 0.01) and higher hatching rates after vitrification (80.0 versus 25.0%, P < 0.05).

Current study assessed differences in the response of sheep to estrus synchronization either by the administration of two doses of prostaglandin or by the insertion of an intravaginal progestagen sponge. The preovulatory follicular dynamics and estradiol secretion, the ovulatory response and progesterone secretion and the number and quality of embryos were studied in 27 ewes treated with two doses of 100 microg of cloprostenol, 10 days apart, and in 29 sheep treated with progestagen sponges for 14 days. Percentage of sheep responding to the synchronization treatments with signs of estrus behaviour was similar between both groups (81.5% versus 72.4%, respectively). The use of progestagen resulted in a higher diameter of the largest follicle (6.6+/-0.2 versus 5.9+/-0.2, P<0.05), and a lower number of small (6.7+/-0.3 versus 9.6+/-0.4, P<0.005) and total follicles (10.3+/-0.3 versus 12.9+/-0.4, P<0.005). However, mean plasma estradiol concentration during the follicular phase was higher in cloprostenol treated sheep (P<0.005). The mean ovulation rate was similar in both treatments (1.7+/-0.2 versus 1.7+/-0.3), but progesterone concentration during the early luteal phase was again higher in sheep treated with cloprostenol (P<0.05). The mean number of retrieved oocytes/embryos was very similar in both treatments (1.2+/-0.2 versus 1.4+/-0.2) and showed similar fertilization rates (70.6% versus 66.7%), but, although differences did not reach statistical significance, final viability rate was higher in cloprostenol than in progestagen treated ewes (58.9% versus 46.1%, P=0.07). Current results give new evidences supporting the negative effects of progestagens on the functionality of ovulatory follicles and support the development of new protocols for assisted reproduction including the use of prostaglandin analogues.

The current study assessed both the effects of in vitro culture and developmental stage of early stage in vivo produced ovine embryos on their ability to survive cryopreservation. Early stage embryos (n=226) were recovered from the oviduct, at different days of the early luteal phase, at three different developmental stages: 2- to 4-cell, 5- to 8-cell and 9- to 12-cell. For each stage, half of the embryos were cultured to the blastocyst stage and frozen thereafter (CF), while the remainder was frozen just after recovery (EF). A third experimental group (BF; n=43) included blastocysts obtained from the uterus and frozen immediately after recovery. Embryo viability post-thawing was determined by assessing their rate of development to the hatched blastocyst stage following in vitro culture. Culture negatively affected embryo viability, since survival rate was higher in blastocysts obtained from the uterus than in those from culture (83.7% versus 66.1%; P<0.05); also the cryosurvival of cultured embryos was lower when the timing of blastocyst formation was extended (P<0.01). However, survival following freezing-thawing of early developmental stages was significantly lower when compared to viability of their counterparts cultured to the blastocyst stage (23.1% versus 66.1%, P<0.0001). In conclusion, our results indicate that, despite the deleterious effects of culture per se, the culture of early in vivo produced ovine embryos to the blastocyst stage prior to be frozen improves their survival after thawing.

A total of 64 ewes was used to determine if the changes in superovulatory yields related to the ovarian status at the start of superovulatory treatment are due to differences in the population of gonadotrophin-responsive follicles, alterations in the processes of ovulation or transport of embryos from oviduct to uterus and/or developmental competence of the oocyte/embryo. Ovarian status at the start of a superovulatory FSH step-down treatment, administered coincidentally with a progestagen, was assessed by ultrasonography. On Day 4 after progestagen withdrawal, embryos were recovered from oviduct and their viability was determined by assessing development in vitro culture (IVC) until the hatched blastocyst stage. In all the ewes, the ovulation rate was related positively to the number of 2-3 mm follicles at first FSH injection (P<0.005). However, the total number of embryos and their viability were related to the more limited category of 3 mm follicles (P<0.05), whereas a higher degeneration rate was related to the number of 2mm follicles. The presence of a corpus luteum (CL) at the start of superovulatory treatment exerted a protective effect on embryonic viability, decreasing the degeneration of embryos. On the other hand, the presence of a dominant follicle at first FSH dose affected the mean size of the pool of follicles responding to the superovulation treatment, because ovulation arose from 3 to 5 mm follicles in absence of large follicles (P<0.05), but from 2 to 3 mm follicles when large follicles were present (P<0.005), indicating atresia in medium sized follicles in the presence of a large follicle.

The administration of growth hormone (GH) or GH plus GnRH antagonists (GnRHa) in sheep allows the enhancement of the pool of gonadotrophin-responsive follicles present in the ovaries and may be useful to increase yields obtained in embryo programmes. The objective of the current study was to evaluate the ability of follicles recruited in response to treatment with GH and GnRHa to grow in response to exogenous follicle stimulating hormone (FSH) and the competence of their oocytes to resume meiosis. Seven females were treated with two doses of GnRHa (days 0 and 3) and three doses of 15 mg of GH (days 3, 4 and 5). Thereafter, this group and a second group (n = 7) were treated with three doses of 1.5 ml of FSH 12 h apart. A third group (control; n = 4) did not receive GH/GnRHa or FSH. The mean number of follicles aspirated on day 7 was higher in ewes treated with GH and GnRHa prior to the stimulation with exogenous FSH than in ewes treated with FSH without pretreatment and in untreated control sheep (20.4 +/- 2.6 vs 17.7 +/- 3.9 and 11.5 +/- 0.8, p < 0.05 and p < 0.01, respectively). The number of recovered cumulus-oocyte complexes after follicular aspiration was higher in the GH/GnRHa + FSH group (8.7 +/- 0.9 vs 6.8 +/- 1.3 in FSH group, n.s., and 4.5 +/- 0.8 in control, p < 0.05), but there were no differences found in the resumption of meiosis (63.1 +/- 9.5% for GH/GnRHa + FSH vs 79.5 +/- 6.3% for FSH and 60.0 +/- 8.8% for control). These results indicate that GH and GnRHa would be useful to increase the number of gonadotrophin-responsive follicles in the ovary, but adjustment of later FSH treatment allowing further development of follicles may be necessary prior to its use in superovulatory protocols.

To test whether inhibin A assays can be used for the prediction of yields in embryo programmes in goats, 50 does were treated with 45 mg FGA sponges (Chronogest) for 16 days plus a single dose of 100 microg i.m. cloprostenol on Day 14, just before the start of administration of eight doses of 1.25 ml of Ovagen twice daily for 4 days. At first FSH injection, the number and size of all follicles > or =2 mm was assessed by transrectal ultrasound and plasma inhibin A levels were measured by specific dimeric assay. There was a positive correlation between number of follicles > or =6 mm (8.8 +/- 0.5) and inhibin A levels at first FSH dose (193.2 +/- 14.5 pg/ml, P<0.05). The mean number of corpora lutea on Day 7 after sponge removal was related to the total number of follicles with a diameter of 2-6 mm at the onset of the FSH treatment (15.3 +/- 0.7, P<0.05). The total number of embryos recovered was related to the number of follicles with 4-6 mm in size (6.2 +/- 0.5, P<0.05) and to the inhibin A levels at first FSH dose (P<0.05). These results suggest that follicles > or =4 mm are the source of inhibin prior to FSH stimulation and are the main source of oocytes resulting in the number of viable embryos recovered after a superovulatory treatment. Hence, the response to superovulatory treatments in goats in terms of the number of embryos can be predicted from either the population of follicles determined by ultrasound or the plasma inhibin A levels at start of the superovulatory FSH treatment.

This review offers an overview of the basic characteristics of in vivo embryo technologies, their current status, the main findings and the advances gained in recent years, and the outstanding subjects for increasing their efficiency. The use of superovulation and embryo transfer procedures remains affected by a high variability in the ovulatory response to hormonal treatment and by a low and variable number of transferable embryos and offspring obtained. This variability has been classically identified with both extrinsic (source, purity of gonadotrophins and protocol of administration) and intrinsic factors (breed, age, nutrition and reproductive status), which are reviewed in this paper. However, emerging data indicate that the main causes of variability are related to endocrine and ovarian factors, and so the number of studies and procedures addressing a better understanding and control of these factors may be increased in the future. The accomplishment of this objective, the improvement of procedures for embryo conservation and for the selection and management of recipient females, will allow further development and application of this technology.

The main objective of this study was to compare the effect of the presence of large follicles at the start of FSH treatment on the superovulatory response in ewes in the breeding and nonbreeding seasons. A second objective was to verify the effect on the superovulatory response of the presence of a corpus luteum at the start of the FSH treatment during the breeding season. Fifteen ewes in breeding season (October) and 14 in nonbreeding season (May-June) were treated with 40 mg FGA sponges (Chronogest) for 14 days, together with a single dose of 125 microg cloprostenol on Day 12, considering Day 0 as day of progestagen insertion. Superovulatory treatments consisted of eight decreasing doses (1.5 ml x 3, 1.25 ml x 2 and 1 ml x 3) of Ovagen twice daily from 60 h before to 24h after sponge removal. Ovarian structures were assessed by transrectal ultrasonography using a 7.5 MHz linear array probe. Luteal activity at progestagen insertion (Day 0) and presence of corpus luteum and of large follicles at first FSH dose (Day 12) were determined. There were no significant differences between the breeding season and nonbreeding season for ovulation rate (11.6+/-1.4 versus 11.6+/-1.3), number of recovered embryos (8.0+/-1.1 versus 9.6+/-1.3) or number of viable embryos (7.2+/-1.1 versus 5.8+/-1.2). During the breeding season, there were fewer recovered embryos in ewes with a large follicle (> or =6mm) at first FSH dose (6.9+/-1.1 versus 12.3+/-1.8, P<0.05) and fewer viable embryos (5.0+/-1.2 versus 10.5+/-0.5, P<0.05) than in ewes without such a follicle. During the nonbreeding season, however, there were no significant differences between ewes with or without a large follicle for either recovered (9.0+/-2.5 versus 11.3+/-1.2) or viable embryos (6.3+/-2.3 versus 8.1+/-1.2). Analysis of seasonal differences in ewes with a large follicle showed a lower number of recovered embryos in the breeding season (P<0.05) due to a lower recovery rate (65.7% versus 92.3%, P<0.05), since mean number of corpora lutea in response to the FSH treatment was similar (10.9+/-1.3 versus 10.0+/-2.5). These results indicate that, in sheep, the inhibitory effects of large follicles during the nonbreeding season are not as obvious as during the breeding season.

Each of sixty Rasa aragonesa ewes received two embryos on Days 2-3 of the estrous cycle (Day 0=estrus) from 27 donors of the same breed that were superovulated with pFSH. The influence of several variables on fertility and prolificacy after transfer was studied by discriminant analysis. Our results showed that the main variables contributing to higher fertility were: in the donor-recipient couple, degree of estrus synchrony between them (better if donors were in estrus before recipients); in recipients, interval from FGA withdrawal to estrus onset, prolificacy in the previous lambing, age (all, better if inferior to the mean) and interval from the previous lambing (better if superior to the mean); in donors, ovulation rate (better if lower than the mean); and in embryos, developmental stage (better if superior to the mean). Likewise, the main variables contributing to higher prolificacy were: in donors, body condition score (better if higher than the mean) and weight (better if inferior to the mean); and in recipients, plasma progesterone concentration at transfer (better if inferior to the mean). The percentage of ewes correctly classified as lambing or not was 71.7% (P<0.01), and 72.5% of the ewes were correctly classified as having one or two lambs (P<0.05). Whether the criteria we have found for optimum results after transfer are applicable or not to conditions other than ours will need to be confirmed.

This study was designed to test the efficiency of recently developed vitrification technology followed by microscope-free thawing and transfer of sheep embryos. In a first set of experiments, in vivo derived embryos at the morula to blastocyst stage were frozen in an automated freezer in ethylene glycol, and after thawing and removal of cryoprotectants, were transferred to recipient ewes according to a standard protocol (control group). A second group of embryos were loaded into open-pulled straws (OPS) and plunged into liquid nitrogen after exposure at room temperature to the media: 10% glycerol (G) for 5 min, 10% G+20% ethylene glycol (EG) for 5 min, 25% G+25% EG for 30s; or 10% EG+10% DMSO for 3 min, 20% EG+20% DMSO+0.3M trehalose for 30s. The OPS were thawed by plunging into tubes containing 0.5M trehalose. After this rapid thawing, the embryos were directly transferred using OPS as the catheter for the transplantation process. In a second set of experiments, in vivo derived and in vitro produced expanded blastocysts were vitrified in OPS and then transferred as described above. The lambing rates recorded (59% for the conventionally cryopreserved in vivo derived embryos, 56% for the vitrified in vivo derived embryos, and 20% for the vitrified in vitro produced embryos), suggest the suitability of the vitrification technique for the transfer of embryos obtained both in vivo and in vitro. This simple technology gives rise to a high embryo survival rate and will no doubt have applications in rearing sheep or other small ruminants.

In a first experiment, embryo viability was estimated after recovery in the uterus or the oviduct of 70 Manchega ewes following a treatment of superovulation with decreasing doses of OVAGEN. Fewer viable embryos (5.6 +/- 0.9 vs. 8.3 +/- 0.8, P < 0.05) and more degenerative embryos (31.3% vs. 6.8%, P < 0.005) were obtained from the uterus than from the oviduct respectively. In a second experiment performed on 14 ewes, embryo viability was analyzed in relation to the follicular population estimated by ultrasonography (follicles > or = 2 mm) at the first FSH administration. Progesterone (P4) and oestradiol 17beta (E2) concentrations were also determined from the beginning of the superovulation treatment to the recovery of the embryos. The number of viable embryos (4.3 +/- 1.4) was positively correlated (r = 0.824) with of 2-4 mm diameter follicles (P < 0.05), and with E2 concentrations at -12 h (r = 0.891, P < 0.01) , 0 h (r = 0.943, P < 0.0001) and +24 h (r = 0.948, P < 0.05) from estrus detection. Prolonged high levels of E2 up to 72 h with low levels of P4 on days 3 and 4 after estrus had a negative (P < 0.05) effect on embryo viability. These results indicate that ovarian response to superovulatory protocols is related to the individual variations in the number of follicles of 2-4 mm at the start of FSH treatment, and that embryo viability is conditioned by the steroid patterns during the time spent in the genital tract of the super-ovulated ewes.