170 ASSESSMENT OF FRESH AND FROZEN–THAWED BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) SPERM DNA FRAGMENTATION USING THE SPERM CHROMATIN DISPERSION TEST
M. J. Sanchez-Calabuig A C , J. de la Fuente A , P. Beltrán-Breña A , E. Martinez-Nevado B , J. F. Perez-Gutierrez C , D. Blyde D , S. Johnston E , K. Harrison F , C. Lopez-Fernandez G and J. Gosalvez GA Departamento Reproduccion Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Madrid, Spain;
B Zoo Aquarium de Madrid, Madrid, Spain;
C Faculty of Veterinary Medicine, Universidad Complutense de Madrid, Madrid, Spain;
D Sea World, Queensland, Australia;
E Wildlife Biology Unit, University of Queensland, Queensland, Australia;
F Queensland Fertility Group, Queensland, Australia;
G Unidad de Genética, Universidad Autónoma de Madrid, Madrid, Spain
Reproduction, Fertility and Development 25(1) 233-234 https://doi.org/10.1071/RDv25n1Ab170
Published: 4 December 2012
Abstract
There has been significant development over the last 20 years to improve genetic management of the captive bottlenose dolphin (Tursiops truncatus) by means of genome resource banking and assisted reproduction. Although standard semen parameters have been analysed in some detail, very little is known about sperm DNA fragmentation (SDF) in this species. The aim of this study was to develop a sperm chromatin dispersion test (SCDt) for the bottlenose dolphin to establish the baseline level of SDF immediately after ejaculation and cryopreservation and to determine the dynamic loss of sperm DNA quality after ex vivo handing and incubation in conditions that mimic the female reproductive tract. Semen from 8 bottlenose dolphins was collected by manual stimulation. Initial validation of the SCDt was conducted by means of in situ nick translation and neutral comet assay using a proven fertile male. To investigate the dynamic loss of sperm chromatin (rate of sDF loss), thawed sperm samples were incubated at 37.9°C for up to 48 h, and aliquots of spermatozoa were assessed after 1, 4, 8, 24, and 48 h. Dolphin sperm nuclei with fragmented DNA exhibited large halos of dispersed DNA fragments, whereas those with nonfragmented DNA displayed small halos of nondispersed DNA. A high correlation (r2 = 0.82; P ≤ 0.01) was found between the respective assessments of the SCDt and the neutral comet assay. All nucleoids resulting in a large halo of dispersed chromatin were intensely positive to in situ nick translation. The level of sDF fragmentation observed immediately after ejaculation in fresh and frozen samples was relatively low (1–5%). After comparing different ejaculates of the same individual, differences were found. Chromatin stability was high during the first 48 h of ejaculation or post-thawing and incubation. Evaluation of the sDF dynamics of fresh and frozen–thawed spermatozoa revealed no significant increase in the baseline level of sDF or in the relative increase of DNA damage after 48 h of incubation. Our data suggest that cryopreservation does not induce a dramatic increase in sperm chromatin damage. Interestingly, sperm samples derived from aged animals resulted in an increased rate of DNA loss, which was observed after 60 min post-incubation.
