Seasonal variations in sperm DNA fragmentation and pregnancy rates obtained after artificial insemination with cooled-stored stallion sperm throughout the breeding season (spring and summer).

The aim of this study was to assess seasonal variations during different periods of the breeding season (spring and summer) on stallion sperm DNA fragmentation and in vivo fertility associated with cooled-stored semen samples. Ejaculates were collected from eleven stallions and assessed for sperm motility (assessed by computer-assisted sperm analysis) and plasma membrane integrity (evaluated under fluorescence microscopy). Sperm DNA fragmentation (evaluated by the Sperm Chromatin Dispersion test) was assessed in cooled-stored semen at 5 °C for up to 24 h. Artificial insemination was performed throughout the breeding season. Mares were inseminated with cooled-stored semen (up to 24 h) every other day until ovulation. Pregnancy rates per cycle were determined detecting the embryonic vesicle by ultrasonography fifteen days after ovulation. Values (mean ± SD) for progressive sperm motility were significantly higher (P < 0.05) in spring (53.57 ± 9.97%) in comparison to summer (41.37 ± 10.81%). No significant differences in plasma membrane integrity were found between seasons (P > 0.05). Sperm DNA fragmentation was significantly lower (P < 0.01) in spring in comparison to summer after 0h (4.81 ± 1.87% vs. 8.77 ± 5.78%), 6h (9.00 ± 3.19% vs. 18.73 ± 8.22%) and 24h (14.6 ± 4.13% vs. 30.14 ± 9.85%) of cooled-storage. Pregnancy rates per cycle were also significantly higher (P < 0.01) in spring (50%) in comparison to summer (37%). There was a moderate negative relationship between positive pregnancies and sperm with fragmented DNA (r = - 0.619; P < 0.001). Semen samples associated with moderate fertility levels (Pregnancy rate < 50%) showed a higher percentage of sperm with fragmented DNA compared to samples obtaining higher fertility levels. In conclusion, seasonal variations were found during the breeding season, obtaining lower sperm DNA fragmentation and higher pregnancy rates in spring. Additionally, samples with the highest proportion of sperm with fragmented DNA showed the lowest fertility levels throughout the breeding season.

Analisis de curvas de fusion de alta resolucion para la tipificacion de la mutacion de la abiotrofia cerebelar equina / High resolution melting analysis for the equine cerebellar abiotrophy mutation typing

INTRODUCCIÓN: la abiotrofia cerebelar equina es una enfermedad neurodegenerativa de etiología genética autosómica y recesiva. Se presenta con más frecuencia en el Pura Raza Árabe. La enfermedad está causada por una mutación puntual que produce una degeneración progresiva de las células de Purkinje. OBJETIVOS: puesta a punto de una técnica sencilla, empleando el análisis de curvas de fusión de alta resolución (HRM), que permita identificar los individuos portadores de la mutación causante de la abiotrofia cerebelosa en los ancestros de un animal enfermo. MATERIAL Y MÉTODOS: extracción de ADN de 93 muestras de sangre provenientes de animales emparentados. Diseño y selección de una pareja de cebadores para la amplificación de una secuencia de 89 pares de bases que contiene la mutación. Amplificación de la secuencia y análisis de las curvas de fusión. RESULTADOS: el análisis genealógico confirmó el carácter autosómico recesivo de la enfermedad. La amplificación no generó fragmentos inespecíficos. La técnica de análisis HRM permitió diferenciar de forma inequívoca los genotipos homocigotos sano y enfermo, y también el genotipo heterocigoto de los portadores sanos. Los resultados coinciden con los obtenidos mediante la técnica de análisis de fragmentos publicada en 2011. CONCLUSIÓN: la técnica desarrollada permite detectar caballos portadores de la abiotrofia cerebelosa de forma sencilla

INTRODUCTION: the equine cerebellar abiotrophy is an autosomal recessive neurodegenerative disease. It has been more frequently described in the Arabian horse. The causative mutation produces a progressive degeneration of Purkinje cells. OBJECTIVES: developing a simple technique based on the high-resolution melt analysis (HRM) in order to identify the carriers of the mutation among a group of ancestors of an affected horse. MATERIAL AND METHODS: DNA extraction of 93 blood samples from a group of related animals. Primers designed to amplify an 89 base pair sequence that contains the mutation. Amplification of the sequence and melting curves analysis. RESULTS: the genealogical analysis confirms the autosomal recessive nature of disease. The amplification did not generate nonspecific fragments. The HRM analysis allowed the differentiation of healthy and affected homozygous genotypes, and also the differentiation of the carrier, heterozygous genotype. Our results concur with those obtained using the fragment analysis technique published in 2011. CONCLUSION: high resolution melting analysis is a simple technique that allows the detection of cerebellar abiotrophy carriers in horses

Evolución de la seroprevalencia de piroplasmosis equina en yeguas del Centro Militar de Cría Caballar de Mazcuerras (Cantabria) entre los años 2010 y 2018 / Seroprevalence evolution of piroplasmosis in mares of the Equine Breeding Military Center of Mazcuerras (Cantabria) between 2010 and 2018

Introducción: La piroplasmosis equina es una enfermedad de declaración obligatoria. Está provocada por dos parásitos diferentes, Theilera equi y Babesia caballi. Los vectores son garrapatas de distintas especies, difiriendo también en sus aspectos clínicos e inmunológicos. Objetivos: Analizar la seroprevalencia de la piroplasmosis equina a lo largo del tiempo, diferenciando los dos agentes etiológicos. Material y métodos: Muestras de suero de yeguas del Centro Militar de Cría Caballar de Mazcuerras (Cantabria) obtenidas en los años 2010, 2014 y 2018. Análisis de anticuerpos en suero mediante enzimoinmunoensayo de competición. Resultados: El estudio muestra que un 84,37% de los animales analizados son seropositivos a uno o a los dos agentes etiológicos de la piroplasmosis equina. Además, se observa un incremento importante de la seropositividad entre el 2010 y el 2014, siendo en 2010 un 65,51%, en 2014 un 95,56% y en 2018 un 89,48%. La seroprevalencia para T. equi ha ido aumentando progresivamente; sin embargo, para B. caballi se observa un aumento entre 2010 y 2014 seguido de un ligero descenso en 2018. Conclusión: T. equi ha incrementado su presencia en las latitudes más septentrionales de nuestro país, reflejado en un progresivo aumento de su seroprevalencia durante los años de estudio en el CMCC de Mazcuerras en Cantabria

Introduction: Equine piroplasmosis is a notifiable disease. It is caused by two different parasites, Theilera equi and Babesia caballi, each of which displays different clinical and immunological features. Their natural vectors are ticks of different species. Objectives: To analyze the seroprevalence of equine piroplasmosis over time, differentiating the two etiological agents. Material and methods: Serum samples from mares from the Equine Breeding Center of Mazcuerras (Cantabria) obtained in 2010, 2014 and 2018. Serum antibody analysis was performed with the enzyme-linked immunosorbent assay. Results: The study shows that 84.37% of the animals analyzed are seropositive to one or both etiological agents of equine piroplasmosis. In addition, a significant increase in seropositivity was observed between 2010 and 2014, from a 65.51% seroprevalence in 2010 to 95.56% in 2014 and 89.48% in 2018. The seroprevalence for T. equi has progressively increased during the studied period. B. caballi only increased from 2010 to 2014, as a slight decrease was observed in 2018. Conclusion: T. equi is increasing its presence in northern latitudes of Spain. This is reflected in the progressive seropositivity increase observed over the years in the Equine Breeding Center of Mazcuerras in Cantabria