Resumen de Estudio del factor térmico en los protocolos de crioconservación del semen de oso pardo: ("ursus arctos")
- español
En esta tesis doctoral se han evaluado diferentes modelos de manejo durante la precongelación de eyaculados de oso pardo, con el fin de obtener un Banco de Recursos Genético eficaz y que sea de aplicación a la población de osos de las montañas cantábricas. Los modelos objeto de estudio han sido: 1)la aplicación de diferentes rampas de refrigeración y la simplificación de los protocolos (eliminación del tiempo de equilibrado sólo o con el periodo de refrigeración); 2) el almacenamiento a corto o largo plazo a 5 °C de las muestras espermáticas; 3) el control de la temperatura, el porcentaje de glicerol o la tasa de dilución, en el almacenamiento a largo plazo; 4) el uso de medio sólido para mejorar la calidad durante el almacenamiento a largo plazo.
- English
Brown bear ejaculates are usually collected under field conditions, so to redefine the classic cryopreservation protocol from this species sperm could be necessary to ship them to reference laboratory for cryopreservation or reproductive biotechnologies (e.g. sex‐sorting) before freezing, which could improve the effectiveness of Genetic Resource Bank of brown bear spermatozoa.
Throughout this work, we assessed different cooling and freezing guidelines of brown bear sperm collected by electroejaculation: 1) cooling rates and simplification of protocols (the omission of equilibration time alone or with cooling period); 2) short or long term storage at 5° C of spermatic samples; 3) control of temperature, glycerol percentage or dilution rate in long‐term storage;
4) the use of solid state to improve sperm quality during long‐term storage.
For assessment the effect of cooling rates and equilibration time before freezing on post‐thawing quality, we tested three cooling rates (0.25, 1 y 4 °C/min), two equilibration times (0 y 1 h) and the omission of cooling and equilibration periods (“direct freezing”) versus standard freezing (cooling at 0.25 °C/min and equilibration for 1 h, control). In first experience, our results indicated post‐thawing scores dropped upon the increase of the cooling rate, rapid cooling rate (4 °C/min) yielded the worst quality scores respect slow one, and both were similar to rate of 1°C/min. Seminal samples kept at 5 °C for 1 hour showed higher results compared with the nonequilibrated ones for both post‐thawing (% dACR) and post‐incubation (% VIAB, TM and PM). Moreover, we observed damage induced in preserved sperm by direct freezing compared with the control group, this damage was more evident after incubation (thermal stress test ‐ThS test‐), for viability, acrosomal status and motility. In conclusion, our results suggest that slow cooling rates (up to 1°C/min) and at least 1 hour equilibration time are necessary for the effective cryopreservation of brown bear sperm.
After this approach about the study of equilibration period, for second esperiments, we addressed, in‐depth, the evaluation of this step; thus, we evaluated different equilibration times (0, 0.5, 1 ‐control‐, 4‐5, 7‐8 y 10‐12 h) and three times for a long‐term storage (24, 48 and 72 h) versus control (1 h), at 5 °C before freezing. In the first experience, no significant differences were found for the different periods of equilibration tested, except for nonequilibrated group (0 h) which showed lower values of iACR, TM and PM. For evaluation of long‐term storage, we observed a decline markedly and progressively since 48 up to 72 h, in post‐thawing quality from brown bear sperm. In view of these results, we suggest that the pre‐freezing cooling period up to 24 h could be extended without freezing the spermatic samples.
For a third step to redefine the classic protocols of brown bear sperm, we analyzed the conditions for long‐term storage, to enable the seminal samples to be transported to reference laboratory. For that, we assessed the effect of three temperatures (room temperature ‐RT‐, 15 y 5 °C), three glycerol percentages (0, 3 y 6 %) and two dilution rate (1:1 ‐1782×106 sperm/mL de media‐ y final ‐a 100×106 sperm/mL), up to 48 h. At 5 °C, samples yielded higher results for VIAB, PM and iACR for 24 hours, and for TM, PM and YOPRO‐ (viable and non‐apoptotic status) after 48 hours, compared with the other temperatures. For glycerol percentage, for 24 h, post‐thawing quality increased progressively with the percentage; after 48 h, 6 % glycerol showed beneficial effects on sperm cryopreservation. Besides, this percentage had a clearly superior freezability (viability and motility). Both dilution rates obtained similar data within first 24 h, but final dilution supported better post‐thawing quality after 48 h. Both dilution rates showed similar freezability, except after 48 h, when the final dilution reached a higher percentage of recovery rates of viability. These findings suggested that the best conditions for long‐term storage (up to 48 h) of brown bear electroejaculates are: a temperature of 5 °C, a glycerol percentage of 6 % and a sperm concentration of 100×106 sperm/mL.
Finally, to improve post‐thawing quality of stored samples for long‐term (up to 48 h), we evaluated the effect of solid state and its handling, so we tested four experimental groups: 1) 1:1 dilution in standard extender at RT (room temperature), cooling in tube, final dilution at 5 °C (Control 1, 24 and 48 h); 2) final dilution in standard extender at RT, cooling in tube (“FD‐Tube”); 3) final dilution in standard extender at RT, cooling in 0.25 mL plastic straw (“FD‐Straw”), and 4) final dilution with standard extender supplemented with 1.5 % gelatine at RT, cooling in 0.25 mL plastic straw (“Gel”). At prefreezing after 48 h of storage, Gel reached higher viability, and progressiveness although lower velocity (VAP). For postthawing analysis, Gel yielded higher VIAB, YOPRO‐, iACR and LIN, but lower VAP and ALH, independently storage time. No differences were found among the rest of experimental groups (Control 24/48 h, FD‐Tube and FD‐Straw). We could conclude that solid state by the supplementation with gelatine, could be a suitable alternative to preserve viability and progressiveness of brown bear spermatozoa stored at 5 °C.
