Banca de QUALIFICAÇÃO: ALINE SARAIVA DE OLIVEIRA

USE OF NANOEMULSIONS OF ESSENTIAL OILS RICH IN LIMONENE AS CONSTITUENTS IN SHEEN SEMEN DILUTORS

semen cryopreservation, nanoemulsions, limonene, surfactant.

The objective was to develop a nanoemulsion of essential oils rich in limonene and to evaluate the influence of their addition to the ovine semen freezing extender on sperm cells. From the emulsification process by means of agitation, an oil-in-water (O/A) nanoemulsion was obtained using citrus essential oils of Sicilian lemon (Citrus limon; NEA) and wild orange (Citrus sinensis; NEB) separately, with soy lecithin as surfactant (LS). For formulation, in the aqueous phase the LS was hydrated in ultrapure water at a concentration of 5% (v/v) in a water bath at 37 °C for at least 60 minutes, and then homogenized at 500 rpm for at least 20 minutes. The oil phase was added to aqueous (1%; v/v) in the form of a slow drip under continuous agitation at 500 rpm. Then, the emulsions were kept under agitation at 1500 rpm for 24 hours to obtain a nanoemulsion and stored under refrigeration until their use. For freezing, ejaculates of six breeders were harvested, evaluated and submitted to the formation of pools (n=7). Then, samples were diluted in Tris-egg yolk (TGO) dilutor for freezing enriched with nanoemulsion at different concentrations (0, 1.5, 2.5 and 3.5%) at the final concentration of 200 x 106 /mL spermatozoa. The samples were filled in straws (0.25 mL), frozen in an automated system and stored in liquid nitrogen (-196 °C). Then, the samples were thawed in a water bath (37 °C/30 sec) and processed for analysis of sperm kinematics (CASA), plasma and acrosome membrane integrity (iMPA) and mitochondrial membrane potential (PMM) parameters by fluorescence microscopy. For all experimental groups, MT was ≥ 30%. In the parameters of sperm kinetics, plasma membrane integrity and mitochondrial membrane potential, no significant difference was observed between the values of the groups supplemented with nanoemulsion (1.5, 2.5 and 3.5%), when compared to the control group. However, the integrity of the acrosome (iAC) showed a significant difference (P>0.05) in the groups treated with NEA at concentrations 2.5 and 3.5% and in all groups of NEB (1.5, 2.5 and 3.5%), thus presenting increase when compared to the control group. It is concluded that the addition of the nanoemulsions of citrus essential oils of Sicilian lemon and wild orange to the freezing extender of sheep semen preserves the integrity of the acrosome membrane of the sperm cells after thawing.