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Post-mortem spermatozoa recovery and freezing in a cantabric brown bear (ursus arctos): A preliminary report
Theriogenology
277 i
i Exotic Species
POST-MORTEM SPERMATOZOA RECOVERY AND FREEZING IN A CANTABRIC BROWN BEAR (Ursus arctos): A PRELIMINARY REPORT L. Anel, F. Martfnez, M. Alvarez, E. Anel, J.C. Boixo, M. Kaabi, P. Paz, C. Chamorro and P. Herr~ez Animal Reproduction. Le6n University. 2407 l, Le6n, Spain. e-mail:[email protected] At present the Cantabric Brown Bear (Ursus arctos), which probably constitutes the last pure breed aggregate of brown bear in the world, is a seriously endangered population (~80 animals are living in a fragmentary area in the North of Spain). The harvesting of gametes obtained post-mortem could be a useful tool in creating a genetic resource bank. The present work is a preliminary report about post-mortem spermatozoa (spz) recovery in a Cantabric Brown Bear (7 years old, 170 kg), died 8 days after an accident in the wilds (3rd May 1998). The testis was extracted 70 min post-mortem and the epididymis was dissected at 5°C. A sample of gametes was obtained from each epididymis region and deferens ductus (reference of the preejaculatory spermatic cells). The percentage and position of the cytoplasmic droplets (CD) were evaluated as a gamete maturity index (viability prediction). Table 1. Cytoplasmic droplets (CD) rates (%) in the epididymal and ductus deferens spz Anterior CD Middle CD Distal CD Without CD Epididymal head 90 2 1 7 Epididymal body 6 82 2 10 Epididymal tail 5 69 8 18 Ductus deferens 6 64 10 20 The results in Table 1 show a similar degree of maturation (percentage of CD types) in epididymal tail and ductus deferens spz. These data (a greater percentage of central CD) suggest, according to other species, that sperm maturation is not complete in this bear. One possible reason may be that the sample was acquired during the non-breeding season. Table 2. Quantitative parameters of the epididymal tail (ET) spermatozoa recovery Epididymis (g) ET (g) Sperm (g) spz/ml Total recovered spz Left 7.10 2.81 0.09 4426x106 929x106 Right 7.81 3.09 0.12 The sperm was obtained by means of several longitudinal cuts in the epididymal tail (results in Table 2) and was diluted into TEST-fructose extender with 20% egg yolk and 4% glycerol. 42 straws (20x10 6 spz/0.25 ml) were frozen in a programmable freezer (-20o C/min) and stored into LN2. A preliminary post-thawing sperm evaluation showed a normal decrease of sperm quality in the thawed sample vs pre-freezing parameters (results in Table 3). Table 3. Diluted pre-freezing and post-thawing sperm characteristics Individual motility (%) Progressive motility (%) Intact acrosomes (%) Pre-freezing 45 30 62 Post-thawing 25 15 29 These results show the need to conduct supplementary studies to improve the post-thawing sperm viability. Further cytophysiological and ultrastrucmral tests in the recovered and postthawed spermatozoa are needed. The results will aid us in selecting the assisted reproductive technique (AI, IVF, ICSI) adequate for using these, so valuable, frozen sperm specimens. This study was supported in part by: Principado de Asturias
277 i
i Exotic Species
POST-MORTEM SPERMATOZOA RECOVERY AND FREEZING IN A CANTABRIC BROWN BEAR (Ursus arctos): A PRELIMINARY REPORT L. Anel, F. Martfnez, M. Alvarez, E. Anel, J.C. Boixo, M. Kaabi, P. Paz, C. Chamorro and P. Herr~ez Animal Reproduction. Le6n University. 2407 l, Le6n, Spain. e-mail:[email protected] At present the Cantabric Brown Bear (Ursus arctos), which probably constitutes the last pure breed aggregate of brown bear in the world, is a seriously endangered population (~80 animals are living in a fragmentary area in the North of Spain). The harvesting of gametes obtained post-mortem could be a useful tool in creating a genetic resource bank. The present work is a preliminary report about post-mortem spermatozoa (spz) recovery in a Cantabric Brown Bear (7 years old, 170 kg), died 8 days after an accident in the wilds (3rd May 1998). The testis was extracted 70 min post-mortem and the epididymis was dissected at 5°C. A sample of gametes was obtained from each epididymis region and deferens ductus (reference of the preejaculatory spermatic cells). The percentage and position of the cytoplasmic droplets (CD) were evaluated as a gamete maturity index (viability prediction). Table 1. Cytoplasmic droplets (CD) rates (%) in the epididymal and ductus deferens spz Anterior CD Middle CD Distal CD Without CD Epididymal head 90 2 1 7 Epididymal body 6 82 2 10 Epididymal tail 5 69 8 18 Ductus deferens 6 64 10 20 The results in Table 1 show a similar degree of maturation (percentage of CD types) in epididymal tail and ductus deferens spz. These data (a greater percentage of central CD) suggest, according to other species, that sperm maturation is not complete in this bear. One possible reason may be that the sample was acquired during the non-breeding season. Table 2. Quantitative parameters of the epididymal tail (ET) spermatozoa recovery Epididymis (g) ET (g) Sperm (g) spz/ml Total recovered spz Left 7.10 2.81 0.09 4426x106 929x106 Right 7.81 3.09 0.12 The sperm was obtained by means of several longitudinal cuts in the epididymal tail (results in Table 2) and was diluted into TEST-fructose extender with 20% egg yolk and 4% glycerol. 42 straws (20x10 6 spz/0.25 ml) were frozen in a programmable freezer (-20o C/min) and stored into LN2. A preliminary post-thawing sperm evaluation showed a normal decrease of sperm quality in the thawed sample vs pre-freezing parameters (results in Table 3). Table 3. Diluted pre-freezing and post-thawing sperm characteristics Individual motility (%) Progressive motility (%) Intact acrosomes (%) Pre-freezing 45 30 62 Post-thawing 25 15 29 These results show the need to conduct supplementary studies to improve the post-thawing sperm viability. Further cytophysiological and ultrastrucmral tests in the recovered and postthawed spermatozoa are needed. The results will aid us in selecting the assisted reproductive technique (AI, IVF, ICSI) adequate for using these, so valuable, frozen sperm specimens. This study was supported in part by: Principado de Asturias