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| Author(s): |
Leonardo Batissaco
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Pirassununga. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD) |
| Defense date: | 2014-10-31 |
| Examining board members: |
Eneiva Carla Carvalho Celeghini;
Rubens Paes de Arruda;
Ticiano Guimaraes Leite
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| Advisor: | Eneiva Carla Carvalho Celeghini |
| Abstract | |
Frozen ram semen has been the subject of many researches, mainly due to the difficulty of cervical transposition during artificial insemination. However, intra-cervical insemination in sheep is often used, resulting in reduced fertility with the use of frozen semen. To this end, this study was divided into two experiments. In the first experiment was verified the potential of cyclodextrin loaded with cholesterol as an additive to the extender in the protection of sperm kinetics, integrity of plasmatic and acrosomal membranes, mitochondrial function, sperm capacitation and production of reactive oxygen species (ROS) in cryopreserved sheep sperm. Five ejaculates from six rams (n = 30) were divided into three treatments: only extender (CON); extender + cyclodextrin loaded with cholesterol (CLC + CHO); and extender + pure cyclodextrin (CLCP). After dilution (50x106 spermatozoa/mL), semen was stored in straws, identified and cryopreserved using an automated system. Two straws from each ejaculate and treatment were thawed (at 37° C for 30 seconds) and analyzed for motility (CASA), morphology of spermatozoa (DIC), plasmatic (PI-H342) and acrosomal (FITC-PSA) membrane integrity, mitochondrial potential (JC-1), production of free radicals (CellRox), lipid peroxidation (BODIPY) and cell membrane permeability (Merocyanine 540). Comparisons between groups were analyzed using PROC MIXED of SAS and the effect of group was detected by Tukey (p <0.05) or by the order of nonparametric statistics (Kruskal-Wallis) test, when necessary. In the 2nd experiment the same ejaculates were divided by freezability based on the total post-thaw motility (TPTM) and the percentage of reduction in total motility (%RTM) when compared fresh and post-thaw semen (only extender), and divided into the groups: high (TPTM ≥ 60% and %RTM ≤ 40%), intermediate (60%> TPTM > 40% and 60%> %RTM > 40%) and low (TPTM ≤ 40% and %RTM ≥ 60%) freezability. CON and CLC + CHO treatments were analyzed within each animal and each group for motility (CASA), plasmatic (PI-H342) and acrosomal (FITC-PSA) membrane integrity, mitochondrial potential (JC-1), lipid peroxidation (BODIPY) and cell membrane permeability (Merocyanine 540). Comparisons were performed by analysis of variance (ANOVA) with a factorial arrangement 3x3 (groups CLC+CHO, CON and fresh X groups high, medium and low freezability), comparisons between groups were analyzed using PROC MIXED of SAS and the group effect was detected by the Tukey test (p < 0.05) or no statistical order parametric (Kruskal-Wallis), when necessary. In the first experimente, CLC + CHO treatment was more effective in preserving the parameters of motility, membrane integrity, and mitochondrial potential compared to other treatments. The CLCP group showed a fall in the parameters of motility, membrane integrity, mitochondrial potential, showing thereby a decrease in sperm preservation. In the 2nd experiment, it was observed that the treatment CLC + CHO showed greater preservation for total and progressive motility, rapid cell preservation, plasmatic and acrosomal membranes and mitochondrial potential when compared to CON. This effect was most significant in the groups of low and intermediate freezability, not being as significant in the group of high freezability. We can conclud from this study that treatment with cyclodextrin loaded with cholesterol contributes to better preservation of sperm parameters in ram semen, especially in animals displaying low freezability, however there where no diference in the high freezability group. (AU) | |
| FAPESP's process: | 12/09927-7 - Cryopreservation of ram semen with incorporation of cholesterol through cyclodextrin |
| Grantee: | Leonardo Batissaco |
| Support Opportunities: | Scholarships in Brazil - Master |