Tesis doctoral de Eva Bussalleu Muntada
The analysis of sperm quality is essential to determine whether seminal doses can be used for artificial insemination (al). Traditionally, these analyses are based on the valuation of sperm motility, morphology, vitality and concentration by light microscopy. Anyway, in recent years, multistaining techniques with fluorchromes have been developed to evaluate the functional status and vitality of spermatozoa from the simultaneous assessment of the integrity of the mitochondrial sheath, the plasmatic membrane, the acrosome and the dna. In this work, a quick and reliable method of staining with fluorochromes was designed for the assessment of the sperm integrity and vitality by marking the mitochondrial sheath with mitotracker®green fm, the acrosome with the lectine trypsin inhibitor from soybean (sbti) fluorochrome conjugated with the fluorochrome alexa fluor ® 488 specific for the proacrosine and, the nucleus with the fluorochromes bis-benzamida (specific for viable cells) and propidium iodide (specific for non-viable cells). this technique gave an estimation of the frequency of spermatozoa with intact plasma membrane, nucleus, mitochondrial sheath and acrosome, and consequently, of the frequency of potentially fertile spermatozoa. This analysis was also highly correlated with the values of motility and morphology obtained using the software sca®producció 2002, and it was equivalent to sperm vitality values obtained using the eosin-nigrosin staining. Apart from designing a fluorochrome multistaining method, in this study, the effect of filtration in different matrixes on the sperm quality of seminal doses from boars with low sperm quality was also evaluated. The sperm quality of seminal doses before and after filtration was evaluated from the analysis of the sperm vitality using the fluorochrome multiplestaining technique, the analysis of the sperm motility, the sperm morphology and the sperm concentration with the software sca®producció 2002, the osmotic resistance, with ort and hrt tests, and the metabolic activity from the production of l-lactate. the filtration of seminal doses from asthenoteratospermic males in columns of sephadex g-25 medium, sephadex g-50 fine, sephadex g-50 medium and sephadex g-75 (with a height of matrix of 10 + 0.5 cm and a flow rate of 1 ml/20 seconds and a working temperature of 23°c in all cases) resulted in an increase in the frequency of mature and viable spermatozoa. Filtration also caused a decrease in the hypoosmotic resistance but not the hypertonic resistance of the spermatozoa, or the sperm motility. Nevertheless, the decrease in sperm motility values was considered transitory, because the metabolic activity and the integrity of the mitochondrial sheath of spermatozoa remained unchanged after filtration. Despite the improvement in the sperm quality, filtration led to a significant drop in sperm concentration. filtration of seminal doses from piétrain boars affected by asthenospermia, asthenoteratospermia and teratospermia in columns of sephadex deae-50 (2.5 + 0.5 cm), sephadex cm- 50 (5 + 0.5 cm), glass wool (2 + 0.5 cm) and glass beads (10 + 0.5 cm), with an outflow of 1 ml/40 seconds and 23 °c of working temperature for all columns produced a significant increase in the frequency of mature spermatozoa without affecting the frequency of motile and viable spermatozoa, or metabolic activity or hypertonic resistance. In contrast the resistance of spermatozoa to hypotonic media decreased significantly. Filtration did not involve changes in the spermatic concentration. in conclusion, the conditions settled for the filtration of refrigerated doses from asthenoteratospermic piétrain boars in neuter sephadex allowed the improvement of the sperm vitality and morphology, a readjustment of filtration conditions like changing the height of the matrix and/or the exit flow rate, is necessary in order to avoid significant sperm loss. Instead, the conditions set for filtration in sephadex ionic, glass wool and glass beads columns are appropriate to enhance the sperm morphology from refrigerated seminal doses from boars wiht low sperm quality used for ia.
Datos académicos de la tesis doctoral «Millora de la qualitat espermÁ tica de dosis seminals de mascles reproductors piétrain per filtració en diverses reÁ¯nes«
- Título de la tesis: Millora de la qualitat espermÁ tica de dosis seminals de mascles reproductors piétrain per filtració en diverses reÁ¯nes
- Autor: Eva Bussalleu Muntada
- Universidad: Girona
- Fecha de lectura de la tesis: 07/03/2008
Dirección y tribunal
- Director de la tesis
- Sergi Bonet Marull
- Tribunal
- Presidente del tribunal: merce Durfort coll
- José álvaro Cebrián pérez (vocal)
- Emilio Martinez garcia (vocal)
- Juan María Vazquez rojas (vocal)