Producció in vitro d'embrions de cabra a través de la técnica d'injeció intracitoplasmática d'espermatozous (icsi): tractament dels gàmetes
- Autores: Irene Menéndez Blanco
- Directores de la Tesis: María Teresa Paramio Nieto (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2020
- Idioma: español
- ISBN: 9788449096334
- Tribunal Calificador de la Tesis: Ana Raquel Jiménez y de Macedo (presid.), María Elena Ibáñez de Sans (secret.), Jan Nevoral (voc.)
- Programa de doctorado: Programa de Doctorado en Producción Animal por la Universidad Autónoma de Barcelona
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX
- Resumen
The development of the intracytoplasmic sperm microinjection technique (ICSI) was a great advance for assisted reproduction laboratories. This technique is very useful in cases of male infertility, cryopreserved gametes and low-quality oocytes. Low-quality oocytes are one of the main factors that negatively affect in vitro embryo production (IVEP) in both humans and animals. In this thesis we use prepubertal goat oocytes as a study model for low-quality oocytes to: 1) Study the effect of cryopreservation of male and female games (Studies 1 and 2) on embryonic development after ICSI and 2) To study the effect of supplementation with a natural antioxidant called Crocetin, during in vitro maturation (IVM) of oocytes, on their embryonic development after ICSI.
In study 1, we compared the use of fresh and frozen sperm for the ICSI technique, evaluating its effect on embryonic development and its relationship with sperm capacitation parameters. The results showed significantly higher level of capacitated spermatozoa in frozen-thawed sample. However, these differences were not translated into differences in pronuclear formation or embryonic development after ICSI between groups.
The second study examined the effect of vitrification on IVM prepubertal goat oocytes on: damage to oocytes assessed by analysis of reactive oxygen species (ROS) and embryonic development with ICSI and parthenogenetic activation (PA). The results showed that the group of vitrified oocytes had higher ROS levels compared to non-vitrified oocytes. After ICSI, the normal formation of zygotes and blastocysts did not show differences between both groups. However, after PA, oocyte division and blastocyst formation decreased significantly with vitrification process.
Study 3 was aimed at improving the competence of prepubertal goat oocytes by supplementing the maturing medium with crocetin, a natural antioxidant. We evaluated the effect of crocetin on molecular and cellular parameters related to oocyte competence (ROS, glutathione (GSH) and mitochondrial activity) and embryonic development after in vitro fertilization (IVF), ICSI, and PA. No significant difference in blastocyst formation was observed between the different concentrations of crocetin 0 µM (control), 0.5 µM, 1 µM and 2 µM after IVF. However, we demonstrate that a 1 µM concentration of crocetin in the maturation medium significantly reduced ROS levels although it did not modify the GSH concentration or mitochondrial activity. Analyzing the effect of crocetin on embryonic development after ICSI and PA, we did not observe any statistically significant difference in the percentage of oocyte division, blastocyst formation or the total number of cells per blastocyst between groups. However, slight improvements such as increased oocyte division and greater blastocyst formation were observed in the crocetin ICSI group whose oocytes where matured with 1 µM crocetin.
In conclusion, we have demonstrated that fresh and frozen-thawed semen can be useful for ICSI of prepubertal goat oocytes without compromising the results. However, with the protocol used, prepubertal goat oocytes after vitrification were not able to develop up to blastocyst after being fertilized by ICSI. The crocetin addition to IVM has not shown significant effects on embryo production from prepubertal goat oocytes in spite of the significant reduction on ROS level and slightly improvement on embryo development after ICSI.
