title: Effect of different concentrations of ascorbic acid on motility, membrane integrity and chromatin status of frozen-thawed canine spermatozoa within six hours of storage at 37 degrees C.
abstract: The aim of this study was to examine comprehensively the effect of ascorbic acid (Asc) on frozen-thawed canine semen. Pooled ejaculates (n = 10) were assessed for quality with a computer-assisted sperm analyser (CASA). After centrifugation, the semen was divided into four aliquots (A-D) of which three were diluted with Uppsala 1 extender (v/v) containing different concentrations of Asc (B: 6.8 mmol/l; C: 13 mmol/l; D: 27 mmol/l). One group without Asc served as control (A). Subsequently, dilution samples were treated and cryopreserved as described previously (Theriogenology 66, 2006, 173). After thawing, samples were stored at 37 degrees C for 1, 3 and 6 h, then examined for quality (CASA, flow cytometry, sperm chromatin structure assay; SCSA). Staining for flow cytometry was performed with FITC-PNA and propidium iodide (Reproduction 128, 2004, 829). The SCSA was performed with both Tris-NACL-EDT buffer (TNE)- and Tris-fructose-citrate buffer (TFC). In the Asc-supplemented groups, percentages of progressively motile sperm (P) were significantly lower than in the control group (A 1 h: 56.6 +/- 9.8, 6 h: 18 +/- 4.5; B 1 h: 51.2 +/- 11.8, 6 h: 13.6 +/- 5; C 1 h: 41 +/- 16.2, 6 h: 11.2 +/- 6.1; D 1 h: 37 +/- 15.2, 6 h: 8.6 +/- 5; p < 0.01), whereas, the percentages of intact cells without acrosome reactions did not differ between groups (p > 0.05). Furthermore, there were no significant differences between TNE- and TFC-samples, for alphaT, for SD of alphaT or for comp alphaT [comp alphaT: TCF-A: 2.5 +/- 1.7%, TNE-A: 3.4 +/- 3.2%; TCF-D: 2.5 +/- 2%, TNE-D: 3.3 +/- 4.3%; p > 0.05]. However, samples diluted with both extenders correlated concerning alphaT, but not comp alphaT. We therefore recommend using TNE-buffer for SCSA with cryopreserved canine semen. In addition, the average alphaT values did not differ significantly between the controls and all other groups (TNE-A: 380.2 +/- 89.1; TNE-D: 338.1 +/- 137.4; p > 0.05). It can be concluded that addition of Asc to cryoextender does not increase quality of frozen-thawed canine semen.
authors.affiliation: Centre for Artificial Insemination and Embryo Transfer, University of Veterinary Science, Vienna, Austria. karineulenberger@gmx.at
authors.forename: K
authors.initials: K
authors.lastname: Eulenberger
authors.forename: S
authors.initials: S
authors.lastname: Schäfer-Somi
authors.forename: C
authors.initials: C
authors.lastname: Aurich
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects*; D002843:Chromatin / Q000187:drug effects* / Q000502:physiology; D015925:Cryopreservation / Q000662:veterinary; D003451:Cryoprotective Agents / Q000494:pharmacology; D004285:Dogs; D008297:Male; D013048:Specimen Handling; D013081:Sperm Motility / Q000187:drug effects*; D013094:Spermatozoa / Q000502:physiology*
chemical_list: D002843:Chromatin; D003451:Cryoprotective Agents; D001205:Ascorbic Acid