2024-03-29T10:32:22Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1425632017-12-18T13:37:42Zcom_10261_108com_10261_8col_10261_361
2017-01-16T13:47:56Z
urn:hdl:10261/142563
Tolerance of brown bear spermatozoa to conditions of pre-freezing cooling rate and equilibration time
López-Urueña, Elena
Martínez-Rodríguez, Carmen
Anel-López, Luis
Paz, Paulino de
Anel, Luis
Ministerio de Ciencia e Innovación (España)
Sociedad Regional Cántabra de Promoción Turística
Sperm cryopreservation
Equilibration time
Direct freezing
Cooling rates
Brown bear
Specific protocols for the cryopreservation of endangered Cantabrian brown bear spermatozoa are critical to create a genetic resource bank. The aim of this study was to assess the effect of cooling rates and equilibration time before freezing on post-thawed brown bear spermatozoa quality. Electroejaculates from 11 mature bears were extended to 100 × 106 spermatozoa/mL in a TES-Tris-Fructose-based extender, cryopreserved following performance of the respective cooling/equilibration protocol each sample was assigned to, and stored at -196 °C for further assessment. Before freezing, after thawing, and after 1 hour's incubation post-thawing at 37 °C (thermal stress test), the quality of the samples was assessed for motility by computer-assisted semen analysis, and for viability (SYBR-14/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate /propidium iodide), and sperm chromatin stability (SCSA) by flow cytometry. In experiment 1, three cooling rates (0.25 °C/min, 1 °C/min, and 4 °C/min) to 5 °C were assessed. After thawing, total motility (%TM) was higher and percentage of damaged acrosomes (%dACR) was lower (P < 0.05) for 0.25 °C/min than for 4 °C/min. The thermal stress test data indicated equally poor quality (P < 0.05) for the 4 °C/min cooled samples in viability (%VIAB), %dACR, %TM, and progressive motility (%PM). In experiment 2, the effect of a pre-freezing equilibration period at 5 °C for 1 hour (cooling at 0.25 °C/min) was evaluated. Samples kept at 5 °C for 1 hour showed higher (P < 0.05) values than the nonequilibrated ones for both thawing (%dACR) and thermal stress test (%VIAB, %TM, and %PM). In experiment 3, samples stored without cooling and equilibration (direct freezing) were compared with the samples cooled at 0.25 °C/min and equilibrated for 1 hour (control freezing). Using thermal stress test, we observed that direct freezing causes damage in viability, acrosomal status, and motility of spermatozoa compared with the control group (P < 0.05). In conclusion, our results suggest that slow cooling rates to 5 °C and at least 1 hour equilibration time are necessary for the effective cryopreservation of brown bear sperm.
2017-01-16T13:47:56Z
2017-01-16T13:47:56Z
2014
2017-01-16T13:47:56Z
artículo
Theriogenology 81(9): 1229-1238 (2014)
http://hdl.handle.net/10261/142563
10.1016/j.theriogenology.2014.02.004
http://dx.doi.org/10.13039/501100004837
eng
Sí
closedAccess
Elsevier