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| Author(s): |
Eneiva Carla Carvalho Celeghini
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD) |
| Defense date: | 2005-05-20 |
| Examining board members: |
Rubens Paes de Arruda;
Rogerio Taveira Barbosa;
Valquiria Hyppólito Barnabe;
Sony Dimas Bicudo;
Cesar Roberto Esper
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| Advisor: | Rubens Paes de Arruda |
| Abstract | |
The process of cryopreservation causes damages in the sperm cellular function which may lead to a reduction in fertility. New techniques to evaluate sperm viability may improve the process of semen evaluation. The present work was divided in two experiments. Experiment 1 aimed the development and validation of practical techniques with high repetibility for simultaneous evaluation of integrity of plasmatic and acrosomal membrane and mitochondrial function of bovine spermatozoa by the strategic association of fluorescent probes. Objective of experiment 2 was to validate the effects of cryopreservation using two differents diluents on sperm motility, plasmatic, acrosomal, mitochondrial membranes integrity and chromatin structure of bovine spermatozoa. A second objective was to verify the correlations between alterations in membranes and motility characteristics in cryopreserved bovine spermatozoa. In experiment 1 propidium iodide (PI, to evaluate plasmatic membrane integrity) and the Pisum sativum agglutinin conjugated with fluorescein isothicyonate- (FITC-PSA, to evaluate acrosomal integrity). Probes were associated to evaluate mitochondrial function in four protocols: Rhodamine 123 (R123, Protocol 1), MitoTracker Green FM (MITO, protocol 2), CMXRos or MitoTracker red (protocol 3), and 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl carbocyanine iodide (JC-1, protocol 4). Results were submitted to analysis of variance, Fishers test and linear regression analysis by the program Stat-View® ( SAS Institute, Inc.1998). It was not possible to validate protocol 1, while the protocols 2, 3 and 4 were validated and showed consistent results. Protocol 4 was used in experiment 2. For experiment 2 seven ejaculates were collected from eight Simental bulls. Volume, concentration, motility and vigour of semen were evaluated subjectively, motility by computer system (CASA), morphological characteristics by differential interference microscopy, plasmatic and acrosomal membranes integrity and, mitochondrial function with FITC-PSA, PI and JC-1 association, and chromatin integrity by acridine orange technique. Then, semen was divided in two aliquots. One was diluted with Bioxcell® and the other with Botu-Bov® diluent, packaged in 0.5 mL straws and frozen using na automated system. Two straws of sêmen from each treatment were thawed and the semen parameter evaluated, as described above. The statistical analysis was performed using the Statistical Analysis System program (SAS Institute Inc., 1985). Data were submitted to analysis of variance and Tukeys test. Pearsons linear correlations were calculated. Cryopreservation increased damages in spermatic structures. Botu-Bov® diluent was better in preserve the motility and membrane integrity (P⁢0,05) when compared to Bioxcell® diluent (AU) | |