title: Evaluation of the effect of cooling and of the addition of collagenase on llama sperm DNA using toluidine blue.
abstract: The effect cryopreservation has on sperm chromatin condensation has been studied in many species but not in South American camelids. The objectives of this study were to evaluate with toluidine blue (TB) the effects of cooling and of adding collagenase on llama sperm DNA condensation. The optimum incubation time (30 s, 1.5 and 3 min) with a reducing agent (dithiothreitol) was also determined. When comparing cooled samples with the raw ejaculate, a significant increase in sperm showing a high degree of decondensation (TB positive) was observed (P = 0.005). A positive correlation was observed, both in raw and cooled semen, between sperm head morphological abnormalities observed in TB-stained cells and TB-positive sperm (highly decondensed DNA), but not with TB-intermediate spermatozoa (moderately decondensed DNA). No significant differences (P > 0.05) were observed in samples incubated with or without 0.1% collagenase. In cooled semen, but not in raw, a significant increase (P = 0.000) in reacted sperm (TB positive) was observed using 3-min incubation with 1% dithiothreitol (DTT). To conclude, cooling would seem to produce an increase in llama sperm chromatin decondensation. Also, 0.1% collagenase in H-TALP-BSA could be added to raw semen to aid its manipulation as it would not seem to increase DNA decondensation.
authors.affiliation: Cátedra de Teriogenología, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires, Argentina. ignaciacarretero@fvet.uba.ar
authors.forename: M I
authors.initials: MI
authors.lastname: Carretero
authors.forename: S M
authors.initials: SM
authors.lastname: Giuliano
authors.forename: C I
authors.initials: CI
authors.lastname: Casaretto
authors.forename: M C
authors.initials: MC
authors.lastname: Gambarotta
authors.forename: D M
authors.initials: DM
authors.lastname: Neild
mesh_terms: D000818:Animals; D002161:Camelids, New World; D002843:Chromatin / Q000378:metabolism; D017364:Collagenases / Q000008:administration & dosage*; D015925:Cryopreservation; D004247:DNA / Q000737:chemistry*; D008297:Male; D012662:Semen Preservation; D013094:Spermatozoa / Q000378:metabolism / Q000648:ultrastructure*; D014048:Tolonium Chloride / Q000737:chemistry*
chemical_list: D002843:Chromatin; D014048:Tolonium Chloride; D004247:DNA; D017364:Collagenases