Evaluating the reproductive ability of breeding rams in North-Eastern Spain using clinical examination of the body and external genitalia

Background Predicting the ability of rams to detect, mate and fertilise ewes in oestrus accurately is certainly difficult; however, tests based on clinical examinations have been performed to assess the overall potential capacity of rams to serve and impregnate ewes. Clinical examinations for breeding soundness evaluation were carried out in 897 Rasa Aragonesa (RA) rams from 35 flocks in North-Eastern (NE) Spain. Clinical examinations of head, trunk, limbs and genitals were performed in each ram. Blood samples were collected for a serological study of Brucella ovis. The sheep owners were surveyed regarding the characteristics of the flock, rams’ health history and the management of rams. The clinical alterations found were classified according to severity (mild or severe). Rams were classified as suitable (without lesions or with only mild lesions) or unsuitable (with severe lesions) for breeding depending on the results of the clinical examinations. Results The results showed that 60.6 % of rams presented some type of alteration (mild: 43.3 %; severe: 17.3 %) in various body parts (genitalia: 31.6 %; head and trunk: 37.2 %; limbs: 15.5 %), and that 16.7 % of rams were classified as unsuitable breeders. The most common genital alterations were ulcerative posthitis (18.7 %) followed by testicular lesions (5.3 %). The highest prevalence of unsuitable breeders was found in the category of adult and aged rams (13.8 % and 37.4 %, respectively) and in the category of emaciated rams (33.3 %). All rams examined were seronegative to Brucella ovis. The mean percentage of rams in flocks was 2.8 % (min: 1.6 %; max: 4.6 %); nevertheless, this percentage dropped to 2.5 % (min: 1.4 %; max: 3.7 %) and 2.1 % (min: 0.3 %; max: 3.5 %) when only suitable or effective (suitable mature) rams were considered. Conclusion Thus, it is concluded that there are fewer effective rams in farms than farmers realise. Frequent clinical examination of males is recommended in order to identify potentially infertile rams.Publishe

New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass

Abstract Background Fungal laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies. Results Firstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λmax of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λmax of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal laccases secreted by yeast. Conclusions The high-throughput screening methods reported in this work could be useful for engineering laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators.This work has been funded by the Spanish National Project EVOFACEL, BIO2010-19697. The internship of X. Chanagá at CIB-CSIC, Madrid, was supported by a grant from the UNAL-Medellín, Colombia. I. Pardo thanks Agencia Estatal CSIC for her JAE-Predoc Fellowship and A.I. Vicente thanks MINECO for her FPI Fellowship.Peer Reviewe

Development of chimeric laccases by directed evolution

28 p.-4 fig.-1 fig.supl.DNA recombination methods are useful tools to generate diversity in directed evolution protein engineering studies. We have designed an array of chimeric laccases with high-redox potential by in vitro and in vivo DNA recombination of two fungal laccases (from Pycnoporus cinnabarinus and PM1 basidiomycete), which were previously tailored by laboratory evolution for functional expression in Saccharomyces cerevisiae. The laccase fusion genes (including the evolved α-factor prepro-leaders for secretion in yeast) were subjected to a round of family shuffling to construct chimeric libraries and the best laccase hybrids were identified in dual high-throughput screening assays. Using this approach, we identified chimeras with up to six crossover events in the whole sequence, and we obtained active hybrid laccases with combined characteristics in terms of pH activity and thermostabilityThis work was supported by the National projects (PIE 200920I207 and BIO2010-19697) and EU Project NMP4-SL-2009-229255.Peer reviewe

Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

15 páginas, 6 figuras, 3 tablas -- PAGS nros. 1370-1384While the Pycnoporus cinnabarinus laccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression in Saccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the α-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved α-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase in kcat. While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (∼23 mg/liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae)This research was funded by EU projects (NMP2-CT-2006-026456, NMP4-SL-2009-229255, COST Action CM0701, and KBBE-2010-4-26537) and national projects (CTQ2005-08925-C02-02, CCG08-CSIC/PPQ-3706, PIE 200920I207, and BIO2010-19697). A.I.C. was supported by a national grant from the Ministerio de Ciencia e InnovaciónPeer reviewe

Transformation of polycyclic aromatic hydrocarbons by laccase is strongly enhanced by phenolic compounds present in soil

8 páginas, 8 figuras -- PAGS nros. 2964-2971Efficient transformation of several polycyclic aromatic hydrocarbons (PAHs) was obtained using a fungal laccase in the presence of phenolic compounds related to those formed in nature during the turnover of lignin and humus. The effect of these natural mediators, namely vanillin, acetovanillone, acetosyringone, syringaldehyde, 2,4,6-trimethylphenol, p-coumaric acid, ferulic acid, and sinapic acid, was compared with that of synthetic mediators such as 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT). Anthracene was significantly degraded by laccase in the absence of mediators, whereas benzoapyrene and pyrene were weakly transformed (less than 15% after 24 h). Vanillin, acetovanillone, 2,4,6-trimethylphenol, and, above all, p-coumaric acid strongly promoted the removal of PAHs by laccase. 9,10-Anthraquinone was the main product detected from anthracene oxidation by all the laccase-mediator systems. The yield of anthraquinone formed was directly correlated with the amount of p-coumaric acid used. This compound resulted in a better laccase mediator than ABTS and close similarity to HBT, attaining 95% removal of anthracene and benzoapyrene and around 50% of pyrene within 24 h. Benzoapyrene 1,6-, 3,6-, and 6,12-quinones were produced during benzoapyrene oxidation with laccase and p-coumaric acid, HBT, or ABTS as mediators, although use of the latter mediator gave further oxidation products that were not produced by the two other systemsThis work has been supported by the Spanish Programmes (CTQ2005-08925-C02-02/PPQ, VEM2004-08559, and BIO 2005-3569) and EU contract NMP2-CT-2006-026456. The authors thank Beldem (Belgium) for P. cinnabarinus laccase preparation and M. J. Vicente and R. Sedano (SIdI, Universidad Autónoma, Madrid) for HPLC/MS, EI, and APCI analyses. S.C. and M.A. thank R&C Contracts of the Spanish MEC, and A.I.C. thanks an I3P Fellowship of CSIC.Peer reviewe

Lacasas de alto potencial redox obtenidas por evolución dirigida

[EN] Method for generating extracellular, functional and soluble laccases having high redox potential and improved activity towards one or more substrates, sequences encoding said laccases, and uses.[ES] Método para generar lacasas de alto potencial redox, extracelulares, funcionales y solubles con actividad mejorada hacia uno o más sustratos, secuencias que codifican dicas lacasas, y usos.Peer reviewedConsejo Superior de Investigaciones Científicas (España), L'intitut National de la Recherche AgronomiqueA1 Solicitud de patente con informe sobre el estado de la técnic

Evolution and current situation of the age at first lambing in Rasa Aragonesa ewes

En ovino, los retrasos en la edad al primer parto dan lugar a periodos improductivos que conllevan una disminución de la vida productiva del animal y un aumento del intervalo generacional, con sus consecuentes pérdidas económicas. Entre los ganaderos no existe una postura única en cuanto a la edad óptima a la que hay que cubrir por primera vez las corderas, en muchos casos por temor a que su estado fisiológico no sea todavía el adecuado y pueda repercutir negativamente en su vida productiva. El peso es uno de los factores más determinantes, ya que la pubertad no aparece hasta que el animal no ha alcanzado un peso vivo y condición corporal adecuados (Foster et al., 1985). En Rasa Aragonesa suele producirse alrededor de los 7-8 meses de edad, aunque puede variar en función de la disponibilidad de alimentos ligada al manejo en la ganaderla. La media de edad al primer parto de los rebaños pertenecientes al programa de selección de UPRAGrupo Pastores en 2015 (19,5 meses; Jurado y Jiménez, 2016) muestra que todavía se está muy lejos del óptimo teórico que se podría alcanzar en esta raza. A pesar de su gran repercusión en la vida productiva del animal, en los últimos años no se le ha prestado la suficiente atención y la mayor parte de los esfuerzos se han dedicado a mejorar otros caracteres como la prolificidad. En este sentido, UPRA-Grupo Pastores viene desarrollando desde 1994 un programa de selección por prolificidad en Rasa Aragonesa. En dicho programa se registran los partos de todos sus animales, contando en la actualidad con más de 2 millones de registros acumulados. Es necesario analizar la evolución de los últimos años y la situación actual de la edad al primer parto en Rasa Aragonesa con vistas a la posible toma de decisiones, lo que constituye el objetivo del presente trabajo.In sheep, delays in the age at first lambing (AFL) give rise to unproductive periods and a decrease in productiva lite. Despite its proven influence on economic profitability, in the Rasa Aragonesa sheep breed the efforts in the last years to improve it have been scarce. The aim of this study was to analyze the evolution of !he las! years and !he curren! state of !he AFL in Rasa Aragonesa ewes far possible luture decision making. Far this purpose 191, 114 fist lambing records from 327 farms were analyzed using the GLM procedure (SAS). Mean AFL during all !he studied period (1997-2013) was 19.8 months, too far from optimal in this breed. A decrease of AFL through !he years was found (-5.6 days/year; P<0.0001). The month of lambing demonstrated an effect on AFL (P<0.0001), so that autumn and winter-born lambs showed the lowest AFL values. The proliftc BMP15 genotype, present in several farms, also gives rise to a slight reduction of AFL (-5 days, P<0.01). The significan! effect of farm on AFL (P<0.0001), with differences up to 421 days, reveals importan! management differences, and therefore, !he great capacity far improvement of AFL in future.Financiado con fondos FEDER a través de POCTEFA (Proyecto PIRINNOVI EFA103/15) y CDTI (Proyecto 101-20160426).Publishe

Age and prolificacy at first lambing of rasa aragonesa ewe lambs carrying or not the FecXr alelle

Desde el descubrimiento en 2007 en la raza Rasa Aragonesa del polimorfismo prolífico Fecx" en el gen BMP15 (Jurado y Calvo, 2007; Martinez-Royo et al., 2008) el número de animales de este genotipo se ha incrementado de manera continua en las ganaderías. Actualmente el 58% (164/283) de los rebaños que forman parte del programa de selección por prolificidad de Unión de Productores de Rasa Aragonesa (UPRA-Grupo Pastores) tienen hembras portadoras heterocigotas (R+), contabilizándose en total 13.024 hembras de este genotipo. De estos, el 38% (63/164) tienen más de un 10% de ovejas R+ (Jurado y Jiménez, 2016). Esto se debe a los efectos demostrados de este polimorfismo sobre la prolificidad de las ovejas heterocigotas y su consecuente impacto positivo en la rentabilidad de las ganaderfas. Las ovejas heterocigotas presentan cambios importantes a nivel de sus follculos ováricos. En concreto, se ve afectada su sensibilidad a las gonadotropinas de manera que son capaces de ovular un mayor número de foliculos con un menor diámetro, y en algunas razas viene acompañado de alteraciones endocrinas relevantes (Fabre et al., 2006). Estos cambios podrfan afectar la entrada en pubertad de los animales. El objetivo del presente trabajo consiste en evaluar la edad al primer parto y la prolificidad de las corderas R+ y ++ en las ganaderlas a lo largo de los últimos 12 años.Since the discovery in 2007 of !he prolific Fecx" polymorphism in !he Rasa Aragonesa sheep breed, !he number of carrier ewes (R+) in farms has significantly increased. These ewes present changes in their ovarían follicles which could lead to endocrine alterations and might affect !he onset of puberty. The objective of !he present work was to evaluate !he age al first lambing (AFL) and !he prolificacy in farm conditions of R+ ewe lambs over !he las! 12 years. AFL was similar in R+ and non-carrier (++) ewes (601 vs_ 600 days), with similar effects of year and month of birth. As expected, prolificacy was higher in R+ comparad to++ ewe lambs (1.47 vs. 1-19; P<0.0001). Over !he studied period AFL significantly decreased and prolificacy significantly increased among years, which demonstrated !he improvements made by sorne farms which perform Al and work with prolific genotypes_ Prolificacy and AFL were related, so that a delay of 365 days in AFL give rise to an increase of 0.21 lambs/lambing ewe lamb, with no interaction with genotype. lt should be taken into account to reach equilibrium between desired age and prolificacy al first lambing.Financiado con fondos FEDER a través de POCTEFA (Proyecto PIRINNOVI EFA103/15) y CDTI (Proyecto IDl-20160426).Publishe