Effects of breed, age, season, and multiple ovulations on cyclic, PGF2α-induced, and postpartum estrus characteristics in Spanish jennies.

This retrospective, population-based, cross-sectional study analyzed data for a total of 104 jennies reared in southern Spain over the period 1995 to 2014. Intervals to ovulation and incidence of multiple ovulation and pregnancy were charted for spontaneous, PGF2α-induced, and postpartum estrous cycles. In spontaneous estrous cycles, the interovulatory interval varied as a function of breed (P < 0.03) and month of ovulation (P < 0.01), and duration of estrus signs was longer in older jennies (0.04). Spontaneous cycles were also associated with higher ovulation rates from September to January (P < 0.006). When PGF2α was used to induce the estrus, not only did estrus signs last longer in old (P < 0.004) and in polyovular (0.02) jennies but old jennies also displayed significantly higher ovulation rates (P < 0.03). In postpartum jennies, no variations were observed as a function of any of the independent variables analyzed. Comparison of ovulation rates between different types of cycle revealed that postpartum jennies exhibited significantly lower ovulation rates (1.32 ± 0.07) and a lower incidence of multiple ovulation (30.4%) than spontaneous (1.62 ± 0.04, 55.0%) and PGF2α-induced (1.74 ± 0.08, 65.5%) groups. No differences were observed in the incidence of ovulation or pregnancy depending on the location of ovulation in polyovular cycles, and ovulation occurred at similar rates in the right and left ovaries. These findings shed further light on reproductive physiology in jennies and may be of value in improving animal management.

Determining ACTB, ATP5B and RPL32 as optimal reference genes for quantitative RT-PCR studies of cryopreserved stallion semen.

Equine germplasm bank management involves not only the conservation and use of semen doses, in addition it can also be a resource to study stallion semen quality and after thawing semen properties for reproductive purposes. A possible criterion to measure quality may be based on differential gene expression of loci involved during spermatogenesis and sperm quality maturation. The rapid degradation of sperm after thawing affects the integrity and availability of RNA. In this study we have analyzed genes expressed in equine cryopreserved sperm, which provided an adequate amplification, specificity, and stability to be used as future reference genes in expression studies. Live spermatozoa were selected from cryopreserved semen straws derived from 20 stallions, through a discontinuous concentration gradient. RNA purification followed a combination of the organic and column extraction methods together with a deoxyribonuclease treatment. The selective amplification of nine candidate genes was undertaken using reverse transcription and real-time polymerase chain reaction (qPCR) carried out in a one-step mode (qRT-PCR). Specificities were tested by melting curves, agarose gel electrophoresis and sequencing. In addition, gene stabilities were also calculated. Results indicated that five out of the nine candidate genes amplified properly (ß-Actin, ATP synthase subunit beta, Protamine 1, L32 ribosomal protein and Ubiquitin B), of which ß-Actin and the L32 Ribosomal protein showed the highest stability thus being the most suitable to be considered as reference genes for equine cryopreserved sperm studies, followed by the ATP synthase subunit beta and Ubiquitin B.

Evaluación de la longitud de los telómeros en caballos, mediante el uso de la reacción en cadena de la polimerasa cuantitativa / Assessment of the length of horse telomeres using the quantitative polymerase chain reaction

INTRODUCCIÓN: Los telómeros son estructuras de los cromosomas eucariotas, que juegan un importante papel en el mantenimiento de la estabilidad e integridad celular, estableciéndose como uno de los posibles biomarcadores del envejecimiento celular en el hombre y otras especies. OBJETIVOS: Evaluar la posibilidad del uso de la técnica de la reacción en cadena de la polimerasa cuantitativa (qPCR) para calcular la longitud relativa de los telómeros en caballos y asociarla con factores como la edad, la raza y el y el sexo. Material y métodos: Se amplificó una secuencia repetitiva correspondiente a los telómeros, junto con otra de un gen de copia única (Interleukina-2), en muestras de sangre de 175 caballos de tres razas, de distintos grupos de edad y de ambos sexos. Se calculó la longitud relativa de los telómeros de cada individuo a través dos variables de respuesta: la diferencia entre el número de ciclos de cuantificación entre telómeros e Interleukina-2 (Dif Cq) y la ratio entre ambos valores (T/S ratio). RESULTADOS: La variable Dif Cq presenta una asociación más fuerte con la edad que la variable T/S ratio. Se observó una correlación negativa entre la edad y la longitud de los telómeros (R= -0,31, p < 0,05). CONCLUSIÓN: La técnica de qPCR es aplicable para determinar la longitud relativa de los telómeros en caballos. Los individuos mayores de 20 años presentan telómeros acortados, aunque no se encontró un efecto significativo ni de la raza, ni del sexo

INTRODUCTION: Telomeres are specialized structures at the ends of eukaryotic chromosomes which play an important role in the maintenance of cell integrity and stability. The telomere length is considered one of the potential biomarkers of cellular aging. OBJECTIVES: To evaluate the possible use of quantitative polymerase chain reaction qPCR in the estimation of relative length of equine chromosome telomeres. MATERIAL AND METHODS: A repetitive sequence corresponding to telomeres and a single copy gene (Interleukin-2) were amplified from blood samples of 175 horses of three breeds, different ages and both sexes. The relative length of telomeres of each individual was calculated by means of the response variables: the difference of quantification cycles number between telomeres and Interleukin-2 (Dif Cq) and the ratio between both values (T/S ratio). RESULTS: The variable Dif Cq has a stronger association with the age than the variable T/S ratio. There was a negative correlation between the age and telomere length (R= -0,31, p < 0,05). CONCLUSION: The qPCR technique is applicable to determine the relative length of telomeres in horses. The length of the telomeres shorten significantly from age 20, although no effect was found over the breed or sex

Selección de genes de referencia en semen equino criopreservado para su uso en estudios de expresión genética con la técnica de PCR cuantitativa / Reference genes selection in cryopreserved equine semen for its use in studies of gene expression with the quantitative PCR technique

INTRODUCCIÓN: La gestión de bancos de germoplasma implica la conservación y uso de dosis seminales, pero también pueden ser una fuente de estudio sobre la calidad de los sementales y las propiedades del semen para su empleo post descongelación. Un criterio para medir la calidad seminal puede basarse en las diferencias de expresión de algunos genes implicados en la espermatogénesis y la maduración espermática. OBJETIVO: Análisis de genes expresados en semen equino criopreservado que ofrezcan una adecuada amplificación, especificidad y estabilidad para su empleo como genes de referencia en futuros estudios de expresión genética. MATERIAL Y MÉTODOS: Purificación de espermatozoides vivos mediante un gradiente de concentración discontinua a partir de pajuelas de semen criopreservado correspondiente a cuatro sementales. Extracción orgánica de ácidos ribonucleicos con tratamiento con la enzima desoxiribonucleasa y la amplificación selectiva de siete genes candidatos mediante retrotranscripción y reacción en cadena de la polimerasa en tiempo real en un solo paso. RESULTADOS: Tres de los genes seleccionados, β-Actina, Ubiquitina B y proteína Ribosomal L32 se amplifican correctamente. β-Actina, Ubiquitina B manifiestan la mayor estabilidad. CONCLUSIÓN: En los espermatozoides procedentes de muestras de semen criopreservado equino se puede detectar la presencia de ARNm, siendo el gen de la β-Actina y de la Ubiquitina B los más indicados como genes de referencia de los siete candidatos analizados

INTRODUCTION: The germoplasm bank management involves the conservation and use of semen doses, but can also be a source of study on the quality of stallions and semen properties for use after thawing. A criterion for measuring the semen quality may be based on differences in expression of some genes involved in spermatogenesis and sperm maturation. OBJECTIVE: Analysis of genes expressed in equine cryopreserved sperm that can provide adequate amplification, specificity and stability for use as future reference genes in gene expression studies. MATERIAL AND METHODS: Purification of live sperm through a discontinuous concentration gradient from cryopreserved semen straws corresponding to four stallions. Organic extraction of ribonucleic acids with deoxyribonuclease treatment and the selective amplification of seven candidate genes using a retrotranscription and a real time chain reaction of the polymerase in one step mode. Specificity is tested by melting curves and agarose gel electrophoresis. Also the stability of the genes is calculated. RESULTS: Three of the selected genes, β-actin, Ubiquitin B and Ribosomal protein L32 were properly amplified. β-Actin and Ubiquitin B showed the best stability. CONCLUSION: mRNA was amplified from equine cryopreserved semen samples, being the β-Actin and the Ubiquitin B genes the most suitable reference genes of the seven candidates analyzed

Protocolo de extracción de ADN en lotes de 10 mosquitos para la identificación de Plasmodium spp. mediante qPCR / Protocol for DNA extraction in batches of 10 mosquitoes for the identification of Plasmodium spp. thorough qPCR

Las tropas que despliegan en zonas de operaciones endémicas de malaria, necesitan de una información precisa del riesgo sanitario para la toma de decisiones acerca de las medidas de prevención más adecuadas. El estado de portador de un mosquito se determina clásicamente por la presencia o ausencia de esporozoitos de Plasmodium spp. en las glándulas salivales. Los protocolos basados en la amplificación del ADN en tiempo real (qPCR) son muy sensibles, sin embargo existen dificultades en la qPCR debido a inhibidores presentes en los tejidos del mosquito, lo que obliga a trabajar de uno en uno. En este trabajo se diseña una qPCR para amplificar una región conservada entre mosquitos de diferentes especies y otros dípteros, con el objetivo de comparar varios protocolos de extracción de ADN y determinar el más eficiente a la hora de procesar lotes de 10 mosquitos (AU)

Troops deployed in operational areas where malaria is endemic, need accurate information on the health risk for this disease to make decisions about the most appropriate prevention measures. The carrier status of a mosquito is classically determined by the presence or absence of sporozoites of Plasmodium spp. in the salivary glands. Protocols based on DNA amplification in real time (qPCR) are very sensitive, however there are difficulties in qPCR due to inhibitors present in tissues of the mosquito, therefore it is necessary to work one by one. In this paper we design a qPCR to amplify a preserved region among different species of mosquitoes and other Diptera, in order to compare various DNA extraction protocols to determine the most efficient one when processing batches of 10 mosquitoes (AU)

Plan de vigilancia entomológica en zona de operaciones. Aproximación al estudio de anofelinos (Diptera: Culicidae) y riesgo entomológico asociado en las bases de Herat y Qala I Naw / Entomological surveillance program in area of operations: approach to the study of anopheline (Diptera: Culicidae) and entomological risk at the bases of Herat and Qala i Naw

Introducción: Cualquier información obtenida para contribuir a la evaluación del riesgo de aparición de brotes de malaria en la fuerza desplegada en zonas de operaciones es de máximo interés para los servicios de medicina preventiva. Tras un estudio piloto de vigilancia entomológica, realizado en la base de Herat (Afganistán) en 2008, se han realizado ahora nuevos estudios protocolizados y con más medios en dicha base, así como en la situada en Qala i Naw (Afganistán). Material y métodos: Entre junio y octubre de 2010 se realizaron muestreos entomológicos en las bases de Herat y Qala i Naw, empleando trampas luminosas de succión tipo mini CDC con luz blanca y ultravioleta, así como una trampa de succión tipo BG-Sentinel de BioGents GmbH con atrayente químico. Las hembras de anofelinos se analizaron mediante la reacción en cadena de la polimerasa (PCR) en busca de su posible infección por Plasmodium spp. Resultados: Se capturaron culícidos pertenecientes a diversos géneros, entre ellos del género Anopheles. Todas las hembras de anofelinos fueron analizadas mediante PCR a tiempo real, siendo todas negativas a Plasmodium spp. Discusión: Entre los ejemplares capturados, Anopheles hyrcanus es un potencial vector de malaria en la zona. Dada su presencia en la base de Herat, habría que valorar la posible existencia de reservorios de Plasmodium spp. en las zonas habitadas cercanas a la misma, y la influencia de factores externos, tales como los vientos predominantes en esa región, que pueden empujar a los mosquitos infectados hacia la base, desde esas zonas. Con los resultados obtenidos se puede establecer un protocolo sencillo de evaluación del riesgo de padecer malaria detectando mosquitos portadores potenciales (riesgo medio) y reales (riesgo alto) (AU)

Introduction: Any information obtained to contribute to the assessment of the risk of malaria outbreaks in the force deployed in area of operations is of greatest interest to preventive medicine services. Following a pilot study of entomological surveillance, carried out at the base of Herat (Afghanistan) in 2008, further protocolised research was recently performed also in Qala i Naw base. Material and methods: Between June and September 2010 an entomological survey took place at the bases of Herat and Qala i Naw (Afghanistan), using for the purpose CDC traps with white and ultraviolet light, as well as a Sentinel trap type with specific chemical attractant BG-Lure®. The females of Anopheles were analysed with the real-time polymerasa chain reaction (PCR) in order to look for the possible infection with Plasmodium spp. Results: Mosquitoes belonging to various genera were captured. All anopheline females were analysed by real-time PCR and all of them were negative to Plasmodium spp. Discussion: Among the captured specimens, Anopheles hyrcanus is a potential vector of malaria in the area. Due to its presence in the base of Herat, we should assess the possible presence of reservoirs of Plasmodium spp. in inhabited areas close to the base, as well as the influence of external factors, such as prevailing winds in this region that could drive infected mosquitoes toward the base from those areas. With the obtained results we could establish a simple protocol to assess the risk of malaria, detecting potential vectors (medium risk) and real vectors (high risk) (AU)

Effects of haemolysis, lipaemia and bilirubinaemia on an enzyme-linked immunosorbent assay for cortisol and free thyroxine in serum samples from dogs.

The possible interference of haemolysis, lipaemia and bilirubinaemia on commercially available enzyme-linked immunosorbent assays (test kits Enzymun-Test; Boehringer Mannheim) for cortisol and free thyroxine (FT4) in canine plasma samples was studied. Serum samples from 20 clinically normal dogs were enriched in vitro with different amounts of fresh haemolysate, lipid and bilirubin and compared with the original sera. The tests were used in connection with the analyser system Enzymum-Test (Boehringer Mannheim) System ES300. Haemolysis significantly (P = 0.039) interfered with the accuracy of FT4 determination independent of haemoglobin concentrations. Thus, haemolysis should be avoided in FT4 testing by competitive enzyme immunoassay. Lipaemia significantly interfered (P = 0.0015) with cortisol determination but not with FT4 determination (P = 0.41). Regression analysis showed that FT4 concentration significantly increased as triglyceride levels increased, especially at concentrations greater than 125 mg dL-1. The positive bias observed was of no diagnostic importance for cortisol results and the increase of FT4 levels could be precisely predicted by linear regression analysis. The addition of bilirubin significantly (P = 0) interfered with cortisol testing but, as with lipaemia, the magnitude of the differences were of no clinical significance.

Validation of a commercially available enzyme-linked immunosorbent assay (ELISA) for the determination of cortisol in canine plasma samples.

Samples were obtained from clinically normal dogs before and after ACTH stimulation and dexamethasone suppression tests. The test kit Enzymun-Test (Boehringer Mannheim) for determining cortisol concentrations in human plasma was used in connection with the analyser system Enzymun-Test (Boehringer Mannheim) System ES300 following the manufacturer's instructions. The intra-assay and inter-assay coefficients of variation were 1.28% and 5.64%, respectively. The mean recovery when assaying samples with a cortisol content of more than 100 nmol/L was 95.41%, but this percentage decreased in samples with lower cortisol levels. The sensitivity of the assay was 2.76 nmol/L. The results of the ACTH stimulation and dexamethasone suppression tests were similar to those published previously. The ELISA method evaluated allows a precise and sensitive determination of cortisol concentrations in canine plasma samples. The major drawback observed was the loss of accuracy at low cortisol concentrations. Since the assay tends then to report lower cortisol concentrations, the generally accepted concentration of 40 nmol/L may not be suitable as the cutoff value in dexamethasone suppression tests.