Photodynamic therapy as an antimicrobial alternative for semen of domestic rooster: analysis of different pre-irradiation time, light irradiance and light radiant exposure

Author(s)

Gabriel Augusto Novaes

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2021-06-28

Examining board members

Ricardo José Garcia Pereira; Denise Calisto Bongalhardo; Martha Simões Ribeiro

• Advisor: Ricardo José Garcia Pereira

Abstract

The poultry industry stands out among different sectors of the Brazilian agribusiness, since Brazil is the third largest producer and the world largest exporter of chicken meat. Therefore, the use of biotechnologies that optimize its productivity is extremely relevant, especially artificial insemination. However, because cloaca is a common route for ejaculate and excreta, high degrees of contamination are often observed in poultry semen. To overcome this situation, many semen extenders include antibiotics in their composition, a practice that intensifies the problem of bacterial resistance. Thus, aiming at generating alternatives for the use of antibiotics, our laboratory started exploring Photodynamic therapy (PDT) for its use in poultry semen. To that end, first we tried to understand how changes in concentration photosensitizer (Methylene Blue MB) and pre-irradiation time with this substance, in the radiant exposure (J / cm2) and irradiance (mW / cm2) of light influence sperm viability and bacteria present in semen. Our results showed that incubation with MB at concentrations below 25 µM does not significantly alter sperm total and progressive motilities, regardless of time (1 or 5 min), but higher concentrations are extremely harmful. Consequently, we decided to start irradiations using an intermediate concentration of MB (10 µM) with the shortest incubation time (1 min), in an attempt to avoid as much as possible. the absorption of MB by sperm cells Irradiations of the seminal samples were carried out using the Led Box equipment 660 nm for 30, 60, 120 and 180s at the irradiance of 44.3, 28.98 and 17.35 mW/ cm2. Data revealed that MB and light by themselves did not influence sperm parameters. However, when they are associated in PDT, whenever times of exposure to light increased, effects on sperm parameters also increased progressively, regardless of the irradiance used. Hence, it was decided in the second phase to fix the shortest exposure times to light (30 and 60 sec.) using three different irradiances, in order to evaluate the antimicrobial efficacy of PDT against the total coliforms and gram-negative bacteria present in the semen of the roosters. Unexpectedly, under these less harmful conditions to sperm, PDT was not able to significantly reduce colonies in the seminal plasma. These findings strongly suggest a failure in the selectivity of MB, which very likely was also being absorbed by sperm cells, which may explain the impact on sperm kinetics and the ineffectiveness in reducing coliforms after irradiation. In view of this, we believe that more in-depth studies in order to better understand the source of the damage caused to sperm kinetics are extremely relevant, as well as research that seeks to increase the selectivity of PDT in relation to coliforms present in seminal plasma, for example investigating new classes of photosensitizers or associating molecules that make MB more selective, for example antimicrobial peptides or nanoparticles. (AU)

• FAPESP's process: 18/02849-7 - Photodynamic therapy as an antimicrobial treatment of domestic rooster's semen: analysis of different pre-irradiation time, light irradiance and light radiant exposure
• Grantee: Gabriel Augusto Novaes
• Support Opportunities: Scholarships in Brazil - Master