242 EFFECT OF SPERMATOZOA HEAD MORPHOMETRIC DIMENSIONS ON FREEZABILITY IN BROWN BEAR (URSUS ARCTOS)

Abstract

Having recently observed that survival of red deer spermatozoa after cryopreservation seemed to reflect the size of sperm heads, we hypothesized that cryoresistance of brown bear spermatozoa might also be dependent on head size, since in a preliminary study we had also observed significant differences in sperm head sizes among male brown bears (median values for area 22.2 µm², perimeter 18.2, length 6.1 and width 4.4 µm). In the present report, we analyzed the post-thaw survival of spermatozoa of 6 brown bears that were assigned to 2 groups (3 bears/group) based on sperm size: Group A with large-size sperm heads; Group B with small-size heads. Ejaculates were obtained by electroejaculation of adult brown bears (semi-free ranging in Cabarceno Park, Cantabria, Spain) 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 in water at 65°C for 6 s and survival was measured. Sperm motility (TM: total, and PM: progressive; %) was assessed microscopically, and sperm viability, acrosome integrity (PI/PNA-FITC), and mitochondrial status (JC-1) were assayed for fresh and thawed sperm by flow cytometry. Recovery rates (RR: thawed/fresh × 100) were calculated for all parameters. For measurement of head size, fresh sperm samples were fixed in glutaraldehyde and slides were air-dried for 2 h. The samples were then stained with Diff-Quik^® staining at 37°C. The area (Ar), perimeter (P), length (L), and width (W) of the heads of >100 spermatozoa per slide were measured (Sperm Class Analyzer^®; Microptic S.L., Barcelona, Spain). Data were analyzed with the SAS ver8 system, and the Wilcoxon test was applied. The respective morphometric dimensions of the 2 groups were practically identical (Ar = 22; P = 18; L = 6; W = 4). The post-thaw recovery rates of spermatozoa from Group A were: TM: 60.1 ± 29.3; PM: 54.8 ± 36.0; viability: 99.4 ± 8.0; acrosomes: 96.2 ± 3.1; mitochondria: 70.9 ± 15.5. The recovery rates for Group B were: TM: 78.7 ± 13.8; PM: 69.0 ± 18.8; viability: 93.8 ± 5.2; acrosomes: 98.2 ± 9.8; mitochondria: 72.5 ± 22.5. Because of the high variability of recovery rates between males within each group, there were no statistical differences between the 2 groups. The absence of differences can be explained by the small number of males examined and the high variability between them. More studies are necessary to determine whether large sperm cells of brown bears are more susceptible to damage during cryopreservation.

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