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| Author(s): |
José de Oliveira Carvalho Neto
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Piracicaba. |
| Institution: | Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC) |
| Defense date: | 2013-06-28 |
| Examining board members: |
Roberto Sartori Filho;
Fernanda da Cruz Landim e Alvarenga;
José Buratini Júnior;
José Antonio Dell'Aqua Junior;
Margot Alves Nunes Dode
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| Advisor: | Roberto Sartori Filho |
| Abstract | |
Sperm sorting can have a great impact on breeding programs because it allows sex selection of the offspring, being its use increased considerably in last few years. Currently the rate of in vitro produced blastocyst using sexed sperm is similar to that obtained with no sexed sperm. However, lower rates of pregnancy and blastocyst are observed when sexed sperm are used for artificial insemination or embryo transfer compared to the conventional sperm. These results suggest that further studies are needed to clarify the effects of sexing process on sperm cells. This study aimed to evaluate molecular and functional changes caused by the sorting process, as well as to identify possible differences between sperm containing the X or Y chromosome. Each ejaculate of Nelore bulls (n=4) was collected and separated into three fractions: non-sexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY), being used in 3 experiments. For the experiment 2 and 3 a fourth group was formed by pooling SX and SY samples (SXY). On the first experiment, methylation pattern of the IGF2 and IGF2R imprinted gene in sexed and non sexed sperm was assessed using sodium bisulfite method. On the experiment 2, we evaluated whether sexing by flow cytometry changes the shape and size of the sperm head, and if these measurements differ between cells that bearing the X or Y chromosome. In the last experiment, the effect of sorting process on sperm longevity during 12 h incubation and their ability to bind to oviduct cells were evaluated. In the experiment related to methylation patterns, sex-sorting procedures have not affected DNA methylation patterns of the IGF2 and IGF2R genes. However, a polymorphism was found in IGF2 gene, and a very specific methylation patterns was observed in the IGF2R gene, probably due to an epigenetic characteristic in Bos indicus cattle. On experiment 2, no differences between X and Y sperm group were observed for any of the studied characteristics. However, using different parameters simultaneously in discriminator analysis, in an attempt to differentiate groups, the greatest discriminator model had 100% accuracy to identify the sperm from NS, SX, SY and SXY group. On experiment 3, it was identified that sorting process affected structural sperm characteristics, inducing a pre capacitation status and acrosome reaction. However, after 12 hours of incubation the kinetics of the sexed sperm was similar to the non sexed sperm. Furthermore, sexing process affected the ability of sperm to maintained bound to the oviduct cells. Regarding X and Y sperm, it was observed that X group had a lower ability to bind to the CTU, being the X sperm more susceptible to sorting process than the Y sperm. These results can contribute to improve the use and results of the sexed sperm, as well as to induce enhancements in the sexing process, or even to develop new technologies for separation of X or Y sperm cell. (AU) | |
| FAPESP's process: | 10/00585-0 - Influence of bovine sperm sexed by flow cytometry in different variables of sperm cells and embryos |
| Grantee: | José de Oliveira Carvalho Neto |
| Support Opportunities: | Scholarships in Brazil - Doctorate |