Optimización de la capacidad fecundante de espermatozoides criopreservados mediante la manipulación de vías de señalización molecular: influencia de la modulación del sistema endocannabinoide y de la aplicación del protocolo SER en equinos y bovinos
The use of reproductive biotechnologies is a cornerstone of the livestock industry. In recent years, in vitro embryo production has shown a steady increase due to advances in the implementation of techniques such as conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI...
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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Ciencias Veterinarias
2025
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_8045 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_8045.dir/8045.PDF |
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| Sumario: | The use of reproductive biotechnologies is a cornerstone of the livestock industry. In recent years, in vitro embryo production has shown a steady increase due to advances in the implementation of techniques such as conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Sperm quality is a key factor for achieving optimal fertilization and in vitro embryonic development rates. Treatments that enhance fertilizing capacity by incubating sperm in appropriate media allow the evaluation of sperm parameters as potential predictors of fertilization rates and embryo quality.
Numerous studies have focused on identifying molecules capable of improving sperm competence for their use in IVF or ICSI techniques.
In equines, conventional IVF shows limited efficiency. Studies reporting the birth of foals through this technique emphasize the need for high-quality sperm samples. In the case of cryopreserved sperm, selection and incubation protocols must be adapted to achieve optimal fertilization rates. For this reason, ICSI remains the technique of choice for in vitro embryo production in equines.
In contrast, in bovines, ICSI is inefficient in achieving adequate fertilization and embryonic development rates. While conventional IVF yields high cleavage rates, its efficiency in supporting embryonic development remains low.
This thesis aimed to optimize the fertilizing capacity of cryopreserved equine and bovine sperm through the manipulation of molecular signaling pathways. In the first chapter, specific incubation conditions were evaluated and established for cryopreserved equine sperm, as the published information for these samples was scarce.
Our results demonstrated that incubating cryopreserved equine sperm in a bicarbonate- and PVA-enriched medium at a pH of 7.5, 38.5 °C, for 45 minutes, induces key molecular events associated with sperm capacitation. These include an increase in cAMP, elevated levels of pPKA (PKA-phosphorylated substrates) and tyrosine phosphorylation (pY), increased intracellular calcium, enhanced hyperactivation parameters, and the occurrence of the acrosome reaction. These conditions supported optimal activation, cleavage, and embryonic development rates following ICSI in porcine, bovine, and equine oocytes.
Additionally, this work characterized, for the first time, the endocannabinoid system in equine sperm, identifying CB1, TRPV1, and GPR55 receptors. Adding anandamide (AEA) to the capacitating medium developed in this study did not significantly affect sperm parameters compared to the use of the medium alone. However, incubating sperm with AEA in a non-capacitating medium increased pY levels. Moreover, the use of a GPR55 receptor antagonist reduced pY levels under capacitating conditions, suggesting a potential role for this receptor in equine sperm capacitation,
though further studies are required to confirm its involvement.
In the second chapter, the effects of the SER protocol (Sperm Energy Restriction and Recovery) were explored. This protocol involves the transient starvation of sperm followed by the restitution of energy sources. In bovine sperm, starvation did not affect motility, which remained active, suggesting the utilization of endogenous energy reserves. Energy restitution after starvation increased total and progressive motility compared to controls.
In equines, starvation ceased sperm motility within 20 minutes, accompanied by a decrease in mitochondrial membrane potential, both of which were restored after the reintroduction of energy sources, alongside an increase in hyperactivated motility. Additionally, in equine sperm, starvation induced an increase in intracellular calcium levels and the percentage of the acrosome reaction, parameters that returned to baseline after energy restitution.
In conclusion, cryopreserved equine sperm requires specific incubation conditions to induce capacitation, and the endocannabinoid system may be involved through the GPR55 receptor. Additionally, the SER protocol showed species-specific effects in bovine and equine sperm, highlighting the metabolic particularities of each species. These strategies offer a promising approach to improving IVF and ICSI protocols, optimizing outcomes in equines and bovines. |
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