Estudio de la congelabilidad de eyaculados de oso pardo (Ursus arctos)
- Autores: Susana Claudia Gomes Alves
- Directores de la Tesis: María Mercedes Álvarez García (dir. tes.), Luis Anel Rodríguez (dir. tes.)
- Lectura: En la Universidad de León ( España ) en 2014
- Idioma: español
- Número de páginas: 147
- Títulos paralelos:
- Study of freezability of brown bear ejaculated (Ursus arctos)
- Tribunal Calificador de la Tesis: Emilio Martínez García (presid.), Julián Garde López-Brea (secret.), Juan María Vázquez Rojas (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: BULERIA
- Resumen
- español
En el presente trabajo se pretende estudiar diferentes aspectos de la congelabilidad de los espermatozoides de oso pardo (Ursus arctos) que permitan desarrollar estrategias de mejora de la calidad post-descongelación. Todo ello como desarrollo de protocolos de criopreservación espermática en el oso pardo como paso fundamental para el establecimiento de un banco de recursos genéticos que constituye una herramienta básica en la conservación de esta especie, seriamente amenazada en España. Para llevar a cabo este estudio, por un lado, se desarrolló un diluyente específico analizando diferentes parámetros relativos a la composición del diluyente seminal y se evaluaron diferentes medios comerciales como alternativa al diluyente específico; y, por otro lado, se estudiaron diferentes estrategias para mejorar la calidad de los eyaculados problemáticos en esta especie (urospermia y espermiolgutinación)
- English
Development of sperm cryopreservation protocols from brown bear (Ursus arctos), is a necessary step on the establishment of a genetic resource bank that appear as a valuable tool on the conservation of this species, seriously endangered in Spain.
The aim of the present work was to study different aspects of brown bear sperm freezability that allow to develop strategies for improving post‐ thawing semen quality.
To develop a specific extender we analyzed different parameters related to the composition of seminal extender and different commercial media were evaluated, as an alternative to the specific extender. Furthermore different strategies were studied to improve the quality of the problematic ejaculates in this species (urospermia and sperm agglutination).
For the development of the specific extender, we evaluated two glycerol concentrations (4 and 8%) and two egg yolk concentrations (10 and 20%), supplement with two additives (Equex paste –surfactant agent‐ and EDTA –chelating agent‐) and two osmolality adjustments of the extender (300 and 320 mOsm/Kg). No differences were found among the two glycerol concentrations studied. This could suggest a certain brown bear sperm tolerance to the cryoprotectant.
With regard to egg yolk concentration, 20% egg yolk showed the highest motility results, percentages of viable spermatozoa and viable spermatozoa with intact acrosome.
Extender supplementation with additives rendered significantly higher results for all studied parameters. For extender osmolality, 300 mOsm/kg showed higher values of average path velocity (VAP), curvilinear velocity (VCL), straight‐line velocity (VSL) and amplitude of lateral head displacement (ALH). We conclude that the most suitable extender to cryopreserve brown bear spermatozoa was Tes‐Tris‐Fructose adjusted to 92 300 mOsm/Kg, supplemented with 20% egg yolk, 4‐8% glycerol and additives (1% Equex paste and 2% EDTA).
As a second step in the strategy of validating effective protocols on brown bear sperm freezing, we evaluated the use of commercial extenders designed to other species (bovine ‐Andromed®, Bioxcell®‐ and canine ‐Triladyl Canine®, CaniPro® and Extender 2‐). The TTF‐ULE/bear extender (Tes‐Tris‐fructose‐egg yolk‐glycerol), specifically developed for brown bear served as control in both experiments. After thawing, total and progressive sperm motility and sperm viability were, significantly, greater for TTF‐ULE/bear and Andromed® than for Bioxcell®. TTF‐ULE/bear also showed better results than Triladyl Canine®, CaniPro® y Extender 2. TTF‐ULE/bear is the most suitable extender for the brown bear semen cryopreservation, but comparable results can be obtained with Andromed®.
Finally, strategies for improving cryopreservation of problematic ejaculates from brown bear were tested. Collection of semen directly into a handling extender ‐TTF‐H‐, resulted in a significant decrease of sperm agglutination in fresh samples, but it had no effect on pre‐freeze and post‐thawing semen quality.
Furthermore, other alternatives were evaluated in order to improve post‐thawing quality of spermatozoa from urospermic ejaculates based on reversing the deleterious effects of urine contamination by applying a pre‐freezing washing with extenders with different osmolality, or use of a sperm centrifugation gradient in pre‐freezing.
With regarding to the first proposal, different pre‐freezing washing treatments (extenders of different osmolality: 200, 300, 400, 500 y 700 mOsm/kg) were tested.
Ejaculates with an initial osmolality of less than 120mOsm/kg treated with prefreezing washing (independently of extender osmolality), and the Control sample 93 showed higher pre‐freezing sperm motility than the raw ejaculate, but sperm viability did not changed.
The samples washed with 700 mOsm/kg solutions showed the lowest prefreezing viability. In the post‐thawing evaluation, pre‐freezing washing treatments did not provide any improvement in comparison with the Control sample (only centrifugation without washing), and treatment with 700 mOsm/kg extender had deleterious effects in all urospermic samples. In summary, the standard freezing protocol provides pot‐thawing results similar to those obtained with the most positive pre‐freezing washing treatments.
Puresperm® density gradient centrifugation applied to urospermic raw semen showed good performance in the cell selection, since selected spermatozoa show an improvement of sperm motility and viability of pre and post‐thawing samples.
The problem of this technique resides in the low quantitative yield on the initially treated cells, issue that is very important considering the high value of the samples.
- español
