PROPOLIS : A NATURAL PRODUCT AS AN ALTERNATIVE FOR DISINFECTION OF EMBRYONATED EGGS FOR INCUBATION

During the cooling process of embryonated eggs, there is a natural air flux from the surface to the inner part of the eggs, carrying contaminants such as bacteria and fungi through the shell’s pores, infecting embryos and resulting in the inability to hatch or poor chick quality. Formaldehyde, a toxic product, is still the most used disinfectant for embryonated eggs in the aviculture industry. In order to evaluate the antimicrobial activity of the green propolis ethanolic extract as an alternative to formaldehyde, 140 hatching eggs from laying hens were collected and submitted to disinfection with five different treatments: T1 without disinfection; T2 formaldehyde fumigated eggs; T3, T4 and T5 disinfection by immersion in propolis solution in the concentrations of 2,400 μg, 240 μg and 24 μg, respectively. The contamination levels by total mesophiles and fungi of the egg shells (Aspergillus sp. and other moulds) after disinfection with propolis were lower than when compared to the control without disinfection. In comparison with formaldehyde, the 240 μg and 24 μg propolis concentrations did not differ regarding antibacterial activity, but for antifungal activity the 2,400 μg and 240 μg concentrations were more efficient. The 2,400 μg and 240 μg propolis treatments presented a hatching rate of 94.1%, compared to only 84.6% for the formaldehyde treatment. The green propolis ethanolic extract presented antibacterial and antifungal activities in embryonated eggs showing that it can be a new natural disinfectant product substituting formaldehyde.


INTRODUCTION
The ideal environment for the embryo development is the same needed for microorganism multiplication.Therefore, contaminated eggs will disseminate microorganisms in incubators and hatchers reducing hatchability and producing low quality chicks (Bramwell, 2000).The practices for keeping the eggs sanitary quality require frequent collection and mainly adequate cleaning and disinfection.During the process of eggs cooling, there is a natural air flux from the surface to inside the eggs which carry contaminants such as bacteria and fungi through shell's pores, infecting the embryo and resulting in the inability to hatch, poor quality chicks or sick birds during growing stage (Scott;Swetnam, 1993;cony et al., 2008).Therefore, the eggs should go as quick as possible through disinfection after being laid, by adequate methods and compounds (SeSti, 2005).
Formaldehyde fumigation method is the disinfectant most frequently used by the poultry industry.Formalin (formaldehyde 40%) is mixed with potassium permanganate, an oxidant agent to generate a gas.The eggs are then exposed to this gas in a closed cabinet or in an adequate room (magraS, 1996).Although this method is efficient in keeping incubation with low levels of contamination and with high levels of hatchability, it is important to highlight that formaldehyde is toxic, not only to birds but also to human beings.Formaldehyde fumigation in pre-incubation causes reduction in the size and number of cells from the tracheal epithelium of embryos and from chicks (Zulkifli et al., 1999;Hayretda;kolankaya, 2008).To human beings, formaldehyde is more dangerous as it is a carcinogen.Nevertheless, it is still used mainly as preservative and disinfectant.The International Agency for Research on Cancer (international..., 2006) classified it as carcinogen due to its association with nasopharyngeal cancer in humans and nasal cancer in rodents.Increase in mortality by lymphohematopoietic neoplasm, especially myeloid leukemia and brain cancer, has been observed in anatomists, pathologists and workers from the funeral industry, for being exposed to formaldehyde (Hauptmann et al., 2009).There are not yet data on the literature related to the risk of cancer in workers from the poultry industry, even knowing that these professionals are constantly exposed to formaldehyde at levels considered above the allowed level (Scott;Swetnam, 1993).Thus, there is a need to search for alternatives to substitute this disinfectant product, especially in aviculture.
Propolis is a resinous substance collected by honey bees from exudates from shoots and flower buds of several plants.It has varied color and consistency and it is used by honey bees to repair honeycombs, to close small openings, to embalm dead insects as well as to protect the hive against invasion of microorganisms (marcucci, 1995).The chemical composition of propolis depends on the biodiversity of the region visited by the honey bees (park et al., 2002).Hence, the substances present in the propolis are directly related to the chemical composition of the resin from the origin plant (caBral et al., 2009).Phenolic compounds, among them the flavonoids, have been considered as one of the main biologically active components from propolis (li et al., 2009), together with cinnamic acid derivatives and its esters and diterpenes (luStoSa, 2008).
Evaluations of the efficiency of disinfectant substances that use natural products are scarce in the literature, especially evaluations for usage in eggs that are incubated.With the aim of evaluating the antimicrobial activity of green propolis, the main objective of this work was to test the use of propolis as disinfectant for embryonated eggs, as substitution for formaldehyde.

Green propolis ethanolic extract
A green propolis ethanol extract at 24% produced by Apis Nativa Produtos Naturais Ltda (PRODA-PYS) and stored at 4º C was used.The propolis was collected in São Paulo state, Brazil.

Eggs disinfection
To evaluate green propolis ethanolic extract antimicrobial activity, 140 eggs collected from 68 weeks old laying hens of 051-Embrapa lineage, from Conjunto Agrotécnico "Visconde da Graça" (CAVG), were used.The eggs were selected and the ones not fit for incubation (dirty, cracked, faulty eggshell and too small or too big) were discarded.The eggs were divided into five treatments with 28 eggs each: T1eggs not submitted to any disinfection process, T2eggs fumigated with formaldehyde 91%, T3, T4 and T5 eggs submitted to disinfection using a propolis solution at concentrations of 2,400 µg, 240 µg and 24 µg respectively, by immersion for a period of 5 minutes (mauldin, 2002).From the total of 140 eggs, 40 eggs (eight eggs from each group) were selected for microbiological analysis at the bacteriology laboratory of the Faculdade de Veterinária of Universidade Federal de Pelotas (UFPel) and the others were incubated for 21 days in the CAVG incubator.On day seven eggs were candled and after hatching, the rate (%) of initial embryonic mortality, fertility and hatchability were determined.

Microbiological evaluation
Microbiological evaluation for determining the contamination level and the eggshell disinfection efficiency was based on the counting of total mesophiles, according to the methodology proposed by Silva et al. (1997).Initially, material from the eggshells was collected with a sterile swab previously damped in 1 mL of sterile saline solution.The swab was placed in a falcon tube with a sterile saline solution and homogenized for 30 seconds.Decimal dilution of the samples in saline solution was performed and aliquots of 0.1 mL from the different dilutions were plated on Standard Count Agar.The samples from eggs, which underwent disinfection process were diluted 10 -1 , 10 -2 and 10 -3 , whereas the egg samples which were not disinfected were diluted 10 -3 , 10 -4 and 10 -5 .Finally, the plates were incubated at 37 ºC for 48 h when the reading of bacterial colony forming unit (CFU/mL) was performed.
In order to evaluate antifungal activity, plating as previously described was also performed, however, the dilution used was 10 -1 and the culture medium was Agar Sabouraud Dextrose.The plates were incubated at 25º C for five days to allow growth of filamentous fungi, where CFU counting for Aspergillus sp., other moulds and total moulds were carried out.
The values of CFU counting were converted into log 10 scale.The variance analysis was performed by General Linear Models -SAS 8.0 (2001) looking for statistically significant differences between the treatments.The variables that presented statistically significant differences to the F test were submitted to Tukey test (P < 0.05) with the aim of identifying differences between the mean of each treatment.

RESULTS
Levels of contamination of the eggshells by total mesophiles expressed in log 10 of CFU/mL after disinfection can be observed in Figure 1.The control treatment differed from the others (P < 0.0001) showing a higher contamination level.However, the propolis treatment in the 240 µg concentration did not differ statistically from the formaldehyde treatment (P > 0.05).
In the evaluation of fungi contamination, Figure 2 shows the contamination by Aspergillus sp., where there was no significant difference between the treatments tested (P > 0.05).Yet, propolis treatments allowed a lower contamination than the control treatment, and the 2,400 µg and 240 µg propolis concentrations afforded a smaller number of colonies than the formaldehyde treatment.In Figure 3 it can be observed the contamination by other fungi, like moulds, in which the treatments with 2,400 µg and 240 µg of propolis concentrations did not differ statistically from the formaldehyde treatment (P > 0.05).On the other hand, these treatments did not differ statistically from the control group.Finally, Figure 4 shows the total fungi contamination (Aspergillus sp. and other moulds).None of the treatments differed statistically from the control group (P > 0.05).
Regarding the embryo diagnosis (Table 1), the control treatment presented higher mortality rate in the first seven days of incubation.The treatments with 2400 µg and 240 µg of propolis concentrations and the formaldehyde treatment did not present mortality in this initial period of incubation.Regarding the eggs hatchability after 21 days of incubation, the propolis treatments (2,400 µg and 240 µg) presented the highest rates (94.1%), even higher than the eggs treated with formaldehyde.The lowest rate was observed in the control treatment (77%).

DISCUSSION
Microbiological analyses are common practices in industrial incubators aiming at detecting total mesophiles and fungi like Aspergillus sp. and other moulds.Considering the fact that the control group did not receive any kind of disinfection, it was expected that this group would present higher microbiological contamination, which happened when the total mesophiles prevalence was evaluated with higher CFU, differing statistically from all the other treatments (P < 0.0001) (Fig. 1).Similar result was observed by cony et al. ( 2008) when they evaluated pulverization and immersion techniques with different disinfectants on embryonic eggs.The three treatments with propolis presented a lower contamination when compared to control group (P < 0.0001), and the treatment with 240 µg did not differ statistically from the treatment with formaldehyde (P > 0.05).This decrease in the level of contamination of eggs makes evident the propolis antibacterial activity.According to Bankova et al. (1999) and marcucci et al. ( 2001), the propolis antibacterial activity is higher against Gram positive bacteria, probably due to flavonoids, acids and aromatic esters present in the resin, which would act on the cell wall structure of these microorganisms through a mechanism not yet elucidated.Other authors have already reported on the propolis antimicrobial activity on a large variety of bacteria, especially on Gram positive (kujumgiev et al., 1999;miorin et al., 2003;uZel et al., 2005).Recently, cardoSo et al. ( 2009) found similar results using green propolis ethanolic extract against Staphylococcus aureus and Staphylococcus intermedius isolates.
Regarding antifungal activity, (Figs. 2, 3 and 4) treatments with propolis allowed a lower contamination of eggs than the control treatment, and propolis concentrations of 2,400 µg and 240 µg were more efficient than formaldehyde.The propolis antifungal (fungistatic and fungicide) activity is attributed to the phenolic acids (cinnamic, feluric and caffeic acids), terpenes and flavonoids like chrysin, ermanina, galangin, kaempferol, pinobanskina and mainly pinocembrine (SiQueira et al., 2009).These substances are found in green propolis (marcucci et al., 2001;cuSHnie;lamB, 2005).It is worth noting that in a study performed by our group (fiScHer et al., 2007) aiming at characterizing the immunomodulator effect of the same green propolis ethanolic extract evaluated in this study regarding its antimicrobial activity, a chromatographic analysis (High Performance Liquid -HPLC) showed high levels of phenolic compounds and cinnamic acid and its derived.In this extract, the flavonoids corresponded to 22.37% of the dried extract (fiScHer et al., 2007).
Through embryo diagnosis it was possible to observe that the eggs that did not go through disinfection resulted in a 25% embryonic mortality rate in the first seven days of incubation.This rate is considered extremely high, as in this initial period of incubation it is accepted rates of up to 3% (roSa; Ávila, 2000).Treatments with propolis in concentrations of 2,400 µg and 240 µg and the formaldehyde treatment did not present mortality in this initial period, indicating that they provided an effective disinfection.When hatchability was evaluated, treatments with propolis were more efficient than the others, what makes evident that in these concentrations, propolis besides having no harmful effect can even increase the eggs hatchability.

CONCLUSIONS
The green propolis ethanolic extract, when used as disinfectant by immersion of embryonated eggs, presented antibacterial and antifungal effect besides not being harmful to the embryo development, allowing high hatchability rates.
The green propolis ethanolic extract is an alternative as a natural product, to the use of formaldehyde for disinfection of embryonated eggs for incubation.

Fig. 1 -
Fig.1-Mean log 10 CFU/mL of total mesophiles from embryonated eggs submitted to disinfection.The control treatment differed from the others (P < 0.0001) by Tukey test.In the others treatments different letters represent statistically significant differences (P < 0.05) by Tukey's test.

Fig. 3 -
Fig. 3 -Mean log 10 CFU/mL of other moulds from embryonated eggs submitted to disinfection.Different letters represent statistically significant differences (P < 0.05) by Tukey test.

Fig. 4 -
Fig.4-Mean log 10 CFU/mL of total moulds from embryonated eggs submitted to disinfection.Different letters represent statistically significant differences (P < 0.05) by Tukey test.

Table 1 -
Embryonic mortality, fertility and hatchability of eggs submitted to disinfection.