Sperm Membrane Functionality in the Dog Assessed by Flow Cytometry

Abstract

The objective assessment of sperm function increases the chances of predicting the fertilizing capacity of a fresh semen sample or diagnosing infertility problems. In this study, the available flow cytometry technique was used to determine the membrane functional capacity of canine spermatozoa. The second fractions of ejaculates from six dogs were pooled, and samples (n = 26) processed to determine the variables: sperm viability and plasma membrane integrity by Sybr-14/Pi staining; phosphatidylserine (PS) translocation by Annexin-V-FITC/PI labelling; acrosome membrane integrity by FITC-conjugated Pisum sativum agglutinin/PI labelling; and mitochondrial membrane potential (ΔΨm) by staining with JC-1. Means for the 26 examined samples indicated that 82.66 ± 2.8% of the viable spermatozoa showed an intact plasma membrane, 8.4 ± 2.6% were moribund, 72.7 ± 16% had an intact acrosome, 80.9 ± 17% had high ΔΨm and 8.1 ± 11% had PS translocation with a PS translocation index of 2.1 ± 3%. Motility was only correlated with PS translocation (R = 0.3901; p = 0.0488), and acrosome membrane integrity was correlated with PS translocation (R = -0.5816; p = 0.0018). This study provides objective physiological data on the functional capacity of canine spermatozoa.

Validación de SYBR-14 y 6-CFDA para Evaluar la Viabilidad e Integridad de la Membrana Plasmática en Espermatozoides Caninos de Raza Chihuahua

“…Nuestros resultados en semen canino de raza Chihuahua fueron inferiores a los obtenidos en pool de semen fresco de perros Pastor Alemán, Golden Retriever y Labrador con un 82,66±2,8% de espermatozoides con membrana plasmática íntegra y 8,4±2,6% de espermatozoides viables con membrana dañada evaluado por citometría de flujo (Cheuquemán et al, 2012). Además, fueron inferiores a los obtenidos mediante microscopía de fluorescencia y en diferentes razas de caninos, con membrana plasmática ínte-gra en un rango de 77-99% (Volpe et al, 2009).…”

Criopreservación de Espermatozoides Caninos a - 80C

“…Solo se colectó la segunda fracción del eyaculado rica en espermatozoides (Kutzler, 2005). Inmediatamente después de la colecta, se realizó la evaluación espermática de la motilidad, concentración por microscopía óptica y funcionabilidad de las membranas espermáticas por citometría de flujo, para asegurar una adecuada calidad seminal (Cheuquemán et al, 2012) como se describe más adelante en el texto (evaluación espermática). Solo eyaculados que presentaron un volumen de segunda fracción > a 1,5 ml, motilidad progresiva >70%, una concentración > a 200 x 10 6 espermatozoides/ml y viabilidad >80% fueron incluidos en este estudio.…”

Customizing Semen Preservation Protocols for Individual Dogs and Individual Species: Sperm Preservation beyond the State of the Art

“…Motility was positively and acrosome membrane integrity was negatively correlated with PS translocation of membranes (Cheuquemán et al. ). However, in a later study by this group, the addition of sperm plasma to the extender (TEY) helped preserve the integrity of plasma and acrosome membranes but seemed to compromise motility in 72 h‐chilled canine spermatozoa (Treulen et al.…”