245 USE OF A TRIPLE STAIN (SYBR-14/PI/MC540) FOR VIABILITY AND CAPACITATION ASSESSMENT IN THAWED SEMEN FROM BROWN BEAR (URSUS ARCTOS)

Abstract

Application of new sperm assessment techniques would improve our capability to determine the effects of cryopreservation on sperm function. This becomes relevant when germplasm banks are established for endangered species, as in the case of the brown bear in Spain. Different triple stain techniques have been used in conjunction with flow cytometry to assess various sperm attributes including viability and acrosome status. However, fluorochromes with similar emission spectra may interfere with data resolution, making acquisition and interpretation of data difficult. The double stain combination of SYBR-14 and PI (propidium iodide; max λ 617) has been widely used to differentiate live from dead spermatozoa (spz), and, more recently, merocyanine 540 (MC; max λ 555) has been used to detect a sperm membrane lipid disorder associated with sperm capacitation. In the present study, we analyzed the suitability of combining SYBR-14/PI with MC for simultaneous determination of the viability and capacitation status of frozen–thawed spermatozoa of brown bears (n = 10; semi-free ranging; Cabarceno Park, Cantabria, Spain) obtained by electroejaculation under general anesthesia (7 mg kg^-1 tiletamine + zolazepan and 2 mg kg^-1 ketamine). Semen was diluted (Tes-Tris-fructose, 8% glycerol, 20% egg yolk, EDTA, and Equex paste), loaded in 0.25-mL straws, and frozen in a biofreezer at 20°C min^-1 to -100°C. After storage in liquid nitrogen, samples were thawed at 65°C for 6 s, divided into 2 aliquots (1–2 million spz mL^-1), extended with 300 µL PBS, and stained with SYBR14 (1.2 µL) and PI (3 µL; LIVE/DEAD^® Sperm Viability Kit; Molecular Probes, Inc., Eugene, OR, USA). To evaluate the possible interaction of MC on sperm viability, half of the aliquots were counterstained with 1.5 µL of MC (diluted with 2.7 µM of DMSO); the other half were not counterstained (control). All tubes were incubated at 37°C for 30 min, and assessed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA). Data were analyzed with Bland-Altman. Results indicated that MC staining was mainly confined to dead spermatozoa (22.2 ± 7.7%), whereas a lower percentage of live spermatozoa (5.7 ± 1.6%; P < 0.05) were also stained with MC. Possibly, the staining of dead spermatozoa with MC was due to capacitation changes induced by cryopreservation. The percentage of live spermatozoa was not different between samples counterstained with MC (68.9 ± 9.2) and non-MC-stained control samples (68.6 ± 8.8). Thus, we consider that MC does not influence SYBR14/PI discrimination of viable spermatozoa, and that the 3 stains can be used simultaneously. However, more studies are necessary to determine whether MC can be used to distinguish the capacitation status of brown bear thawed spermatozoa.

This work was supported by CANTUR S.A. and CICYT (CGL 2004-0278/BOS).