Nanoformulações com extrato de Spirulina platensis para utilização na criopreservação de espermatozoides caprinos

Abstract

Goat farming products have been increasingly sought after in recent years. To keep up with this interest, biotechniques need to be used to maximize production and improve product quality. Artificial insemination (AI) is the most widely used biotechnique. In conjunction with it, cryopreservation is used to facilitate and reduce the cost of the process. However, cryopreservation results in cryoinjuries to the sperm during the process. Therefore, diluents are used to protect the cells during the temperature drop. However, current diluents are of animal origin and can promote contamination and disease transmission, in addition to being difficult to standardize. Given this, the objective of this study was to formulate a diluent with Spirulina platensis (Sp) extract for the cryopreservation of goat sperm. Initially, the compounds of Spirulina platensis (D9Z) were extracted using an ethanolic solvent. Next, these compounds were characterized based on analyses of Total Phenolic Content (TPC), Antioxidant Activity (DPPH, FRAP, and ABTS), Determination of Reducing Sugar Content, Fourier Transform Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance Spectroscopy (NMR), and Fatty Acid Profile by Gas Chromatography (GC). In this first stage, we can observe the presence of antioxidant compounds with phenolic results (19.04 mg GAE. g−1) and antioxidants (DPPH: 133.10 μmol Trolox. g−1; FRAP: 143.59 μmol Trolox. g−1; ABTS: 136.02 μmol Trolox. g−1), in addition to reducing sugars (DNS: 0.25 mg/mL). With the aid of FTIR and NMR, we were able to note the presence of possible compounds, such as free amino acids and short peptides, carbohydrates and polyols, fatty acids and unsaturated lipids, phenolic compounds, and pigments such as phycocyanin or β-carotene. GC showed the presence of D-lactic acid, butanedioic acid, L-proline, pentadecane, palmitelaidic acid, hexadecanoic acid, linolenic acid, 9-12-octadecadienoic acid, nonacosane, tetracosane, and triacontane. The presence of these compounds shows the complexity of the extract obtained in our work and its potential use in addition to the cryopreservation diluent, since there are components that help the cell during the temperature drop, such as antioxidants to reduce reactive oxygen species (ROS), sugars that help with energy support, and provide support outside the cell. Furthermore, the presence of other compounds, such as fatty acids, promotes cell membrane protection. As a result, this extract has the potential to be used in the diluent to support the cell during the stages of the process. Subsequently, nanoemulsions were prepared with phosphatidylcholine, extract (ESp), and/or medium-chain triglycerides (MCT) or polaxamer 188 (P188): MC (micelle), NE1 (1% lecithin + 50 μg/mL ESp), NE2 (1% lecithin + 1% MCT), NE3 (1% lecithin + 50 μg/mL ESp + 1% MCT), NE4 (1% lecithin + 50 μg/mL ESp + 1% P188), NE5 (1% lecithin and 1% MCT + 1% P188), and NE6 (1% lecithin + 50 μg/mL ESp + 1% MCT + 1% P188). These groups were analyzed by Zetasizer Nano ZS90 for size, polydispersity index (PDI), and zeta potential and used in the formulation of the diluents. The nanoemulsions obtained an average between 172.0 nm (NE6) and 211.8 nm (NE2), while the MC reached 211.6 nm. The latter also had a PDI of 0.3960, while the nanoemulsions showed values between 0.1687 (NE3) and 0.4123 (NE4). Finally, the micelle presented a zeta potential of -16.13 mV, while the others ranged between -10.12 mV (NE6) and -17.89 mV (NE1). Although these values varied among themselves, they did not show destabilization. Given this, we moved on to the next stage, where the experimental groups for the formation of the diluents were then assembled: GC (micelle), G1 (50% GC + 50% NE1), G2 (50% GC + 50% NE2), G3 (50% GC + 50% NE3), G4 (50% GC + 50% NE4), G5 (50% GC + 50% NE5), and G6 (50% GC + 50% NE6). Initially, they were evaluated for osmolarity, viscosity, and antimicrobial activity. Next, ejaculates from breeding goats were homogenized and subjected to the freezing protocol using the seven experimental groups. After thawing, the semen samples were evaluated for kinetics (Computer Assisted Sperm Analysis), plasma membrane integrity, and mitochondrial membrane potential. The osmolarity of the groups ranged from 1034 (G1) to 1137 (G6). This variation was low, in addition to maintaining an ideal parameter for goat cell diluents. As for viscosity, an increase was noted with the drop in temperature in the groups with the presence of polaxamer 188. This contributes to the characteristic of this surfactant, which gels with decreasing temperature. Furthermore, this effect was not observed in the same way in the groups without the presence of P188, showing that the greater increase is due to the addition of polaxamer to the formulation. In the antimicrobial evaluation, it was observed that most treatments promoted, to some degree, the inhibition of microbial growth, presenting bactericidal/fungicidal or bacteriostatic/fungistatic effects. Thus, we can construct a diluent with SP extract without the need to add antibiotics to the medium. Finally, in the analyses with sperm cells, there was no statistical difference between the MC, G1, G2, G3, and G4 groups in all analyses, showing that replacing half of the phosphatidylcholine micelle with nanoemulsions did not generate a negative effect and maintained the parameters when compared to MC, which may be related to the composition of the added SP extract. However, a significant difference (p<0.05) was observed in G5 and numerically in G6 compared to the other groups in most assessments, showing the negative effect of the synergy between the concentration of MCT, the presence of P188, and the concentration of glycerol. Based on the above, the components present in the extract have the potential to protect cells by lowering the temperature; however, the concentration of the extract must be adjusted for better performance.