Oxidative stress is defined as an imbalance between the production of oxidants and antioxidants. The induction of stress tolerance in oocytes leads to a better embryonic development. In cattle incubating mature oocytes with different stressors (thermal, high hydrostatic pressure, oxidative) increase the generation rate of blastocysts. The purpose of this study was to evaluate the effect of modulating the redox state increasing the oxidative stress through H2O2 in mature oocyte under in vitro culture conditions and its effect on the potential of embryonic development. To do this, oocytes from slaughterhouse ovaries were matured in TCM-199 medium supplemented for 2223 h at 38.5 °C, 5 % CO2 and humidified atmosphere. At the end of 2223 h, the treatments with 0, 50, 100 and 200 µM H2O2 were applied for 1 h. IVF was performed co-incubating the eggs for 18 h with a final concentration of 1x106 sperm/mL. The presumptive zygotes were denuded and cultured in medium KSOM-0.4 % BSA to 38.5 °C in an atmosphere of low concentration of O2 (5 % O2, 5 % CO2 and 90 % N2) and humidified atmosphere. The results show that the induction of oxidative stress by H2O2 produces a similar effect using a concentration of 50 and 100 mM in the cleavage rate of embryos compared to control (88.7 %, 83.2 % and 86,4 % respectively, p>0.05) and decreasing significantly by using a concentration of 200 mM (58.8 %, p<0.05). Also, H2O2 caused a similar effect on the rate of blastocysts with 50 µM (20.4 % vs. 25.8 control, p>0.05) but decreased significantly with 100 and 200 µM (10.7 % and 3.3 % respectively, p<0.05). It is possible that these embryos resistant to oxidative stress may have a higher survival in the cryopreservation processes that generating high levels of reactive oxygen species.

El estrés oxidativo es definido como un desbalance entre la producción de oxidantes y antioxidantes. La inducción de tolerancia a estrés en los ovocitos conllevaría a un mejor desarrollo embrionario. En bovinos, la incubación de ovocitos maduros con diferentes estresores (térmicos, alta presión hidrostática, oxidativos) incrementaría la tasa de generación de blastocitos. Este estudio evalúa el efecto de la modulación del estado redox incrementando el estrés oxidativo con H2O2 en ovocitos maduros bajo condiciones de cultivo in vitro y su efecto sobre el potencial de desarrollo embrionario. Para ello, ovocitos procedentes de ovarios de matadero fueron madurados en medio TCM-199 suplementado durante 2223 h, a 38,5 °C, 5 % CO2 y humedad a saturación. Al final de las 2223 h se incubaron los ovocitos maduros con 0, 50, 100 y 200 µM H2O2. La fecundación in vitro se realizó co-incubando los ovocitos durante 18 h con una concentración final de 1x106 espermatozoides/mL. Los presuntos cigotos fueron denudados y cultivados en medio KSOM-0,4 % BSA a 38,5 °C en atmósfera de baja tensión de O2 (5 % O2, 5 % CO2 y 90 % N2) y humedad a saturación. El estrés oxidativo inducido con H2O2 a una concentración de 50 y 100 µM produce una tasa de división de los embriones similar al control (88,7 %, 83,2 % y 86,4 % respectivamente, p>0,05), disminuyendo significativamente al utilizar una concentración de 200 µM (58,8 %, p<0,05). Asimismo, H2O2 causó un efecto similar en la tasa de blastocitos con 50 µM (20,4 % vs. 25,8 % control, p>0.05) pero disminuyó significativamente con 100 y 200 µM (10,7 % y 3,3 % respectivamente, p<0,05). Es posible, que estos embriones resistentes al estrés oxidativo puedan tener una mayor sobrevida durante los procesos de criopreservación que generan altos niveles de especies reactivas de oxígeno en los embriones.

Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

Since the first successful somatic cell nuclear transfer (SCNT) experiments were carried out, a number of domestic and agriculture species have been cloned using donor cells derived from different sources and origin. However, differences in nuclear transfer efficiency both in vitro and in vivo have been generally observed. These differences may be accentuated when transgenic cell lines are used as nuclear donors in an attempt to generate transgenic cloned offspring. The present study examined the suitability of cell lines derived from 3 different fetal sources and the effects of genetic manipulation of donor fetal fibroblasts with a red fluorescent plasmid, on the in vitro developmental potential and quality of nuclear transfer derived bovine embryos. We observed no differences in the cleavage rate of nuclear transfer embryos generated with any of the cell lines evaluated. However, the blastocyst rate was significantly affected when cell lines were derived from the 3 different fetal sources (21, 18 and 11%, respectively) or from 2 transgenic clonal cell lines that had originated from the same primary fetal cell (18 and 10%, respectively). Despite this difference, quality of embryos as measured by the total number of cells and by assessing some morphology aspects of their appearance was not different. Together these results indicate that fetal fibroblast cell lines derived from different fetal sources and transgenic clonal cell lines that had originated from the same fetus results in different in vitro developmental potential when used as donors for nuclear transfer experiments. Further studies, including evaluation of pregnancy rates, development to term, and epigenetic modifications of these cell lines will be necessary to better understand the differences observed in nuclear transfer efficiency.

Um total de 25 isolados do vírus da diarréia viral bovina (BVDV), sendo 15 originarias do Peru e 10 do Chile foram sujeitas a caracterização genética. A árvore filogenética baseada na análise da região proximal não-codificante (5'UTR) do genoma viral demonstrou que as 25 estirpes pertencem ao genótipo 1 do vírus BVD. Vinte e três destas estirpes puderam adicionalmente ser subdivididas no subtipo 1b, enquanto duas das 10 estirpes isolados provenientes do Chile foram identificadas como pertencentes ao subtipo 1a. Em conclusão, 23 de um total de 25 isolados analisados pertencem ao genótipo 1, subtipo 1b. Este é o subtipo de BVDV predominante em muitos países do mundo, incluindo os EUA. A elevada similaridade genética com isolados descritos anteriormente em outras regiões do mundo realça o papel do comércio internacional de gado no estabelecimento de diversidade genética do vírus BVD. Tanto o Peru como o Chile têm historia de importação de grandes quantidades de gado dos Estados Unidos e da Europa, no entanto sem suficiente documentação comprovativa do estado sanitário no que concerne a esta virose.

Twenty-five BVDV strains, detected in serum from persistently infected cattle from Peru (n=15) and Chile (n=10) were genetically characterized. The phylogenetic analysis based on the 5' UTR showed that all 25 strains belonged to genotype 1. Twenty-three of the strains could further be subdivided into subtype 1b, and two out of ten Chilean strains into subtype 1a. In conclusion, in total 23 out of 25 strains analyzed were of genotype 1, subtype 1b. This is the predominant BVDV subtype in many countries all over the world, including USA. The close homology with previously described strains reflects the influence of livestock trade on the diversity of BVDV circulating within and between countries and continents. Peru and Chile have imported large numbers of cattle from USA and Europe, mostly with insufficient or lacking health documentation.

Animal products traceability has acquired considerable importance as a security measure in EEC member countries since the food crisis of the mid-nineties. This has led to reinforcing the capacity to manage cattle product quality, with traceability emerging as the main tool to prevent risks to product security and quality as demanded by consumers in developed countries. The practical application of a traceability system for beef, based on molecular markers requires the election of a panel of microsatellites, as well as the optimization of methods of sampling and DNA analysis. In this work, a traceability system for beef based on a panel of 10 microsatellites markers was implemented. Different biological samples were evaluated, such as hair, blood, tissue and meat. Hair samples were the most suitable because they are easy to obtain and to manipulate, and have a low storage cost; whereas in the food processing chain, meat samples were the most suitable due to the facility of obtaining from the maturation room. The traceability system was evaluated in a meat processing plant, confirming traceability of 150 samples of hair with their respective meat counterparts with a 100% of certainty, demonstrating the reliability of the developed method. The implemented system is an important contribution since it allows for ensuring the quality of animal products, and can be used as a tool to certify conventional traceability systems. This would allow for increasing the competitiveness of this sector and generating greater confidence among consumers.

La trazabilidad de productos de origen animal ha adquirido gran relevancia como un elemento de seguridad ante las crisis alimentarias ocurridas a partir de mediados de los noventa en países de la Comunidad Europea. Esto ha reforzado la gestión de calidad en productos pecuarios, surgiendo la trazabilidad como la principal herramienta para disminuir los riesgos de seguridad y calidad de los productos que demandan los consumidores de países desarrollados. La aplicación práctica de un sistema de trazabilidad de la carne bovina basado en marcadores moleculares, requiere la elección de microsatélites y optimizar los métodos de muestreo y análisis de ADN. En este trabajo se implementó un sistema de trazabilidad de carne bovina basado en un panel de 10 marcadores microsatélites. Se evaluaron diferentes muestras biológicas como pelo, sangre, tejido y carne de la canal, encontrándose que las muestras de pelo fueron las más idóneas por su facilidad de obtención, manipulación y costo de almacenamiento, mientras que la carne de la canal fue la contramuestra más adecuada, pudiendo obtenerse fácilmente en el frigorífico. El sistema se evaluó en una planta de procesamiento de carne, confirmando con un 100% de certeza la trazabilidad de 150 muestras de pelo con sus respectivas canales, demostrando la confiabilidad del método desarrollado. El sistema evaluado puede ser un importante aporte al sector pecuario del país pues permite garantizar el origen de los productos bovinos y puede ser utilizado como herramienta certificadora de la trazabilidad convencional, aumentando la competitividad y generando mayor confianza en el consumidor.

Total number of cells in cloned embryos is generally lower than that of in vivo derived embryos and in bovines cell allocation at the blastocyst stage, has been observed to be affected in a large proportion of cloned embryos. The current embryo staining procedures are toxic for mammalian cells and thus can not be used to determine the developmental potential of a stained embryo. Therefore, in the present study we sought to assess the feasibility to develop a noninvasive embryo model that would be suitable for the evaluation of cloned embryos subjected to different nuclear transfer and embryo culture procedures. For doing this, we stably transfected a bovine embryonic fibroblast cell line and generated a number of clones that constitutively expressed a red fluorescent protein (HcRed) in the nuclear compartment of the cell. Those clones with normal chromosomal content were further used as nuclear donor in nuclear transfer procedures (SCNT) to generate transgenic cloned embryos. These embryos expressed the red fluorescent protein in each blastomere, allowing their in vivo evaluation during development, thus demonstrating the potential of this model as a noninvasive tool for the assessment of the quality of cloned embryos.

The feasibility of ablating differentiated adipocytes and the mechanism of cell ablation with a suitable prodrug activating system is described. The system is based on the use of E. coli nitroreductase (NTR) enzyme that activates certain nitro compounds, such as the antitumor drug CB1954, into cytotoxic DNA interstrand cross-linking agents. Differentiated preadipocyte cells (3T3L1) transfected with an aP2 driven nitroreductase construct were efficiently killed after incubation with medium containing the prodrug CB1954, while untransfected cells were not affected. It was demonstrated that the mechanism of cell ablation is apoptosis and that the system has a bystander effect mediated by a toxic metabolite of the prodrug. The described system should provide a good alternative approach for gene therapy studies and a new inducible approach to manipulating the number of cells in tissues of transgenic animals and the ability to study the recovery of the tissue from cell damage or loss

The adhesion deficiency of bovine leukocytes to antigens, known as BLAD (bovine leukocyte adhesion deficiency), is a hereditary genetic disease which is lethal for the Holstein breed. It is a recessive autosomal disease that can be transmitted to the offspring. The objective of this research was to standardize the molecular techniques to diagnose BLAD and also to get a first approximation of the genetic frequency of the mutated allele in the bull population of the IXth and Xth Regions. DNA amplification using polymerase chain reaction (PCR) and posterior digestion with restriction enzymes Taq I and Hae III was performed. The digested product was analyzed by 4% agarose gel electrophoresis and ethidium bromide staining to show the typical restriction fragments present in normal, carrier and infected cattle. The technique was validated with a screening test of 59 cattle. Out of 55 bulls analyzed, one turned out to be a BLAD carrier, which suggests a genetic frequency of 1.79% in the bull population of the IXth and Xth Regions. The diagnostic technique of PCR and the digestion of the amplification product with two restriction enzymes (Taq I and Hae III) has identified with accuracy the genotype of cattle at a specific locus and allows an early identification of the genetic defect known as BLAD in infected and carrier animals.

La deficiencia en la capacidad de unión de leucocitos bovinos a los antígenos, más conocida como BLAD, es una enfermedad hereditaria que resulta letal para el ganado de la raza Holstein. Su principal característica es ser una enfermedad autosómica recesiva que puede ser transmitida a la descendencia. El objetivo del trabajo fue estandarizar la metodología para realizar un diagnóstico de la enfermedad mediante técnicas moleculares y lograr una primera aproximación sobre la frecuencia génica del alelo mutado en algunas poblaciones de toros de la IX y X Región. En base a la amplificación del ADN mediante reacción en cadena de polimerasa (PCR) y posterior digestión con enzimas de restricción Taq I y Hae III, fue posible visualizar, mediante electroforesis en gel de agarosa al 4%, los fragmentos de restricción característicos para bovinos normales, portadores o enfermos. La técnica se validó mediante un muestreo de 59 bovinos. De los 55 toros analizados uno resultó ser portador de BLAD, lo que implica una frecuencia génica de 1,79% en la población de toros de la IX y X Región. La técnica de PCR acoplada a la digestión con enzimas de restricción (Hae III y Taq I) ha demostrado que identifica inequívocamente el genotipo del ganado en un locus determinado y permite el diagnóstico certero y precoz de aquellos animales enfermos y/o portadores del defecto hereditario conocido como BLAD.