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| Author(s): |
Vinicius de Seixas Queiroz
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD) |
| Defense date: | 2003-11-28 |
| Examining board members: |
Marcelo Alcindo de Barros Vaz Guimarães;
Valquiria Hyppolito Barnabe;
Maria Denise Lopes
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| Advisor: | Marcelo Alcindo de Barros Vaz Guimarães |
| Abstract | |
This study aimed to investigate the effect of ocelot semen refrigeration on Sperm Motility Index [SMI=(%M+PMx5)/2; %M = proportion of motile spermatozoa ; PM = Progressive Motility], acrossomal integrity (AI) and sperm capacitation. Another objective was to evaluate the FITC-PNA/IP technique efficacy on evaluating simultaneously sperm viability (SV) and AI. Five ocelots, were electroejaculated, the semen was evaluated and only ejaculates (n=16) presenting %M>=60% and PM>=3 were used. Sperm AI was evaluated using Fast Green / Rose Bengal staining (FGRB). The ejaculates were diluted 1:1 in Platz Diluent Variant and subjected to the transportation protocols: Room Temperature and Cooling, -0.23ºC/min, (experiment 1); or only Room Temperature (experiments 2 and 3). After 2 hours, the aliquots were rewarmed and samples were taken to re-evaluate the parameters observed before the transport. The spermatozoa were washed in Hams F10 medium, ressuspended in fresh medium and processed differently, according the experiment: (1) after pre-incubation (38ºC; 5%CO2) during 0, 1, 2 and 4 hours, samples were taken at each time point to be incubated in the absence and presence of 1mM calcium ionophore A23187 (Ca2+Ion), SMI and AI were evaluated; (2) after pre-incubation during 0, 1 and 2h, aliquots were incubated in the absence and presence of 1 and 2 mM Ca2+Ion; SMI and AI were evaluated; (3) after pre-incubation during 9h, aliquots were taken every hour to compare the evaluation of SV and AI (a) separately by the FGRB staining and SMI or (b) simultaneously by the FITC-PNA / IP technique. Cooling caused decline (p<0.02) on AI (71.0%) and SMI (67.1), when compared to values observed before transportation (88.5%; 85.4). Maintenance at room temperature didnt affect (p>0.1) these variables (84.8%; 76.4). Among cooled samples, spermatozoa exposed to Ca2+Ion showed smaller (P<0.01) AI value (52.4%) compared to the group incubated without that substance (55.56%). For samples transported at room temperature, it wasnt observed difference (P>0.05) between the groups with and without ionophore (64.41% vs. 63.87%). When time intervals were analysed separately, the only treatment in which there was effect (p<0,05) of Ca2+Ion on AI was the group refrigerated and pre-incubated for 2h. There was a reduction (p<0,05) on SMI and AI due simply to incubation, even in the absence of Ca2+Ion. The 2µM concentration of this substance was more effective to induce acrosome reaction than 1µM. FITC-PNA and IP fluorocromes bound spermatozoa at the expected sites. However, proportion of marked cells varied randomly during pre-incubation, and didnt correlate (p>0,1) with SMI. IA evaluated by FGRB staining showed positive correlation (r=0,77; p<0,0001) with SMI, decreasing (p<0,0001) during incubation. Cooling was disadvantageous compared to maintaining semen at room temperature, since it was deleterious to spermatozoa membranes and function, and made those cells capable to answer the Ca2+Ion challenge, a characteristic observed in capacitated spermatozoa. Ca2+Ion induced acrosome reaction assay must be improved to allow accurate evaluation of sperm capacitation on ocelots. FGRB staining associated to SMI evaluation was more efficient and easier to perform, than FITC-PNA/IP technique, for AI and SV investigation. (AU) | |