Can Propolis, the Natural Disinfectant of Bees, be Used As an Effective and Healty Disinfectant for Hatching Eggs?

As the first and most important stage of raising poultry, incubation management is an essential factor in profitability (Hulet, 2007). With today’s high-capacity incubators, even the smallest problems during the hatching process may cause significant financial loss (Cadirci, 2009; Kamanlý et al., 2009). Bacterial contamination in eggs begins before ovulation and continues thereafter. The shell of a freshly formed egg contains 300 to 500 bacteria on average. The bacterial load of the egg may change rapidly depending on the environment of ovulation and post-ovulation storage conditions. In particular, eggs laid on the ground can get contaminated with feces and this number may rise to 20.000 to 30.000 in one hour (Elibol et al., 2003). This causes a drop in hatchability rates by increasing the number of embryo deaths. Moreover, it is a cause of omphalitis in chicks, leading to poor survival rates and developmental retardation (Cadirci, 2009; Ernst et al., 1980). Even though reducing the mechanical contamination of eggs has a positive effect on the outcome of incubation, it is required to disinfect the eggs to improve hatchability and maximize the number of healthy chicks (Cadirci, 2009; Ibrahim et al., 2018). Fertilized egg supply from different breeders and transfer of incubated chicks between farms are also factors facilitating spread of disease, further stressing the importance of disinfecting eggs (Elibol et al., 2003).
        
Disinfection process employs oxidized water and chemicals such as sodium hydroxide, phenols and hydrogen peroxide (Baylan et al., 2015). Another method most commonly used in the disinfection of hatching eggs, but at the same time subject to most heated debates is formaldehyde fumigation. Formaldehyde is an excellent anti-microbial agent, but also a toxic chemical and can damage the embryo. In recent years, alternative natural products are used to wipe, spray or immerse eggs in order to control microbial contamination and reduce or eliminate the dependence on synthetic pesticides (Baylan et al., 2015; Cadirci, 2009). Some studies show that formaldehyde is a cause of respiratory, neurological, reproductive and digestive systems diseases in humans, as well as of some types of cancer (Lam et al., 2018).
        
Propolis is a glue-like sticky substance collected by honey bees from leaves, buds, branches and tree barks. Its color ranges from dark yellow to brown and bees use it to polish and disinfect hive interiors and honeycomb cells as a protection against microbial infections. It contains an abundance of vitamins and minerals and has antiseptic, anti-inflammatory, antioxidant, antibacterial, antimycotic, antifungal, antiulcerative, anticarcinogenic and immunomodulatory effects. Because of these properties, it is used both for research and treatment in various medical fields (Akpinar et al., 2015; Aygun and Sert, 2013; Aygun et al., 2012; Sarýçoban and Yerlikaya, 2016).
        
In light of this information, in this study hatching quail eggs were immersed in 5% and 10% propolis, in 0.1% formalin and purified water in separate groups before the incubation and stored for three different lengths of time in order to assess the influences of these factors on the hatch outcomes and shell microorganism counts. The propolis extract used in the study was supplied by a manufacturer employing the method of dissolving organic propolis in water. It is known that propolis is traditionally dissolved in alcohol in such studies and no literature was found on the use of water-based propolis. 

Hatching Eggs Used
 
The study used hatching eggs collected for 2 days from the multi-level hatching cages in the Poultry Unit of the Atatürk University Food and Livestock Application and Research Center. In each cage were 1 male and 3 female breeding quails (Coturnix coturnix japonica) all at the same age (18 weeks) and fed ad libitum with bird food containing 20% protein and 2900 kcal/kg metabolic energy.
 
Preparing the Solutions for Treatment
 
Fanus Gýda® supplied the 10% water-based organic propolis extract used in the study. This product was mixed with sterile distilled water to obtain an extract in the ratio of 5%. Since water was used as solvent in these two experiment groups, the effect of water was evaluated with distilled water in another group. Moreover, a formalin solution of 37% was mixed with sterile water to obtain a 0.1% formalin solution.
 
Experimental Applications
 
A total of 480 fresh Coturnix coturnix (Japanese quail) eggs were used in the study. These were divided into 5 treatment groups as control, distilled water, 5% propolis, 10% propolis, and 0.1% formalin and the eggs in each group were randomly distributed further into 3 groups in an environment of 15 to 18°C temperature and to be collected after 7, 14, and 21 days respectively. 32 eggs were used in each subgroup. Weight loss was calculated on the basis of weekly egg weighing according to storage period. Following the storage, eggs were pre-warmed in a room at 25°C for 6 hours and incubated in a preliminary development machine (with a temperature of 37.5°C, a humidity 65% and turning the eggs at 1 hour intervals) for 15 days. At the end of the 15^th day, the eggs were weighed again and transferred to the output section (temperature 36.5°C, humidity 75%, no turning).
        
Once the hatched chicks dried, they were counted and weighed. Eggs that did not hatch were broken to identify the unfertilized and the ones with a dead embryo, the times of death were also specified for the latter. The eggs were evaluated in 3 groups according to the death of embryos: early term (1-6 days), medium term (7-14 days) and late term (15-18 days). In order to determine the outcome of incubation, 132 eggs found to be unfertilized were left out during the evaluation and (early-, medium- and late-term) embryo mortality was calculated as hatchability (number of chicks/number of fertilized eggs) and percentage (%). 
 
Microbial Analyses
 
For the identification of microbial activity 100 eggs were used in addition to the eggs used in hatching, in a total of 5 groups (4 different treatments and 1 control) and total counts of aerobic-mesophilic bacteria, E. coli, yeast and mold and S. aureus were achieved on day 0, in week 1, week 2 and week 3.
 
Preparing the dilution
 
A dilution of 1/10 was prepared by soaking 1 egg in 50 ml sterile ringer water. Other dilutions were prepared with the dilution method (Kurt et al., 1996).
 
Total aerobic-mesophilic bacteria count
 
Plate Count Agar (PCA, Merck) medium was used to count the microorganisms in this group and plates were evaluated following an incubation of 72+1 hours at 30 ± 1°C (Harrigan and McCance, 1976).
 
Yeast and mold count
 
Rose Bengal Chloramphenicol Agar (RBC, Merck) medium was used for the yeast and mold count. After incubating the petri dishes for 5 days at 21°C, the microorganism count was performed (Harrigan and McCance, 1976).
 
S. aureus count
 
Baird Parker Agar (BPA, Merc) medium was used for counting this microorganism. The count was performed following the incubation of petri dishes at 30°C for 24-48 hours (Harrigan and McCance, 1976).
 
E. Coli count
 
Tryptone Bile X-glucuronide Agar (Merck 1.16122) was seeded with a suitable dilution of 0.1 with the spread plate technique, followed by a 48-hour incubation at 44°C. Blue-green colonies were evaluated (Halkman and Sagdas, 2005).
 
Statistical Analysis
 
The study was carried out according to the fully randomized block trial plan. SPSS software package was used to analyze the data obtained in the study (SPSS and Inc.). One-Way ANOVA was used to calculate egg weight loss, chicks’ hatching weight and microbial analyses; treatment groups and the storage times were calculated with the Logistic Regression method; Chi-Square was used for embryonic deaths and finally, the T-test procedure was applied to find the effect of egg weight loss to hatching.

Egg weight loss
 
Egg weight loss during the incubation period was not affected by the treatment method (P>0.05). However, the eggs stored for 3 weeks lost more weight in the pre-development period than those stored for a 1- and 2-week period and therefore they weighed less prior to the transfer to hatching (P<0.01, Table 1). Some researches on the subject it was found that egg weight loss during the storage (Aygun and Sert, 2013; Aygun et al., 2012; Akpinar et al., 2015) and incubation periods (Aygun and Sert, 2013; Shahein and Sedeek, 2014) were lower in the group with propolis as compared to other treatment groups.
 

        
Weight loss rates of hatching and non-hatching eggs were 0.172 ± 0.04 and 0.147 ± 0.01 g, respectively and it was found that the rate of weight loss had a negative effect on hatching (P<0.01, Table 2). Moreover, it was found that the greater the weight loss, the smaller was the hatching ratio. Study showed that eggs stored for 3 weeks were 4 times less hatchable (P<0.01). Suggested cause is that extended storage times affected internal quality of the egg. Various other studies support this result and suggest that long-term storage affects the internal quality of eggs negatively. Further weight loss during incubation resulted in a higher number of late-stage embryonic deaths in quail eggs, in addition to reducing hatchability (Jones and Musgrove, 2005; Lacin et al., 2008; Toplu et al., 2007). In another study, eggs stored for 1, 2, 3 and 4 weeks had a hatchability of 60.2%, 57.9%, 42.4% and 16.2% respectively and it was reported that the longer was the storage time, the lower was hatchability (Wilson, 1984).
 
  
 
Table 3 shows the logistic regression results of treatment groups and storage times as per hatching. Results show that treatment was not a significant factor affecting chick output or hatchability (P>0.05). However, a highly significant correlation was found between the storage period and hatchability and it was determined that a 3-week storage decreased hatchability almost 4 times (P<0.01, Table 3). In some studies using water (Simsek and Bayraktar, 2005), propolis (Aygun et al., 2012) and other disinfectants (Bailey et al., 1996) it was determined that the treatment method did not affect hatchability. In other studies using propolis in different percentages, it has been reported that the treatment method affects hatchability (Ýbas, 2018; Shaheen and Sedeek, 2014).
 

 
Hatchability
 
Table 4 shows hatchability (number of chicks/number of fertilized eggs) by treatment group and storage time. Upon comparison of hatchability between 1- and 2-week storage and treatment groups, it was found that in both storage times, control groups returned the lowest rate of hatchability.
 
  
 
No correlation was observed between treatment methods and embryo deaths (P>0.05, Table 5). Similarly, in two different studies examining how disinfection of hatching quail eggs with propolis in different percentages and the other treatment method affected the microbial load and incubation parameters, reported no difference between applications in terms of embryonic mortality rates (Aygun et al., 2012; Aygun and Sert, 2013). However, in some other studies, propolis groups were found to be effective on embryo death (Shahein and Sedeek, 2014; Ýbas, 2018).
 
  
 
In the current study, the highest rate of embryonic death was observed in eggs stored for 3 weeks, but the storage period did not affect embryonic deaths (P>0.05). In another study, early-term embryonic death rate in eggs stored for 14 days was found higher as compared to eggs stored for 7 days and it was concluded that extended storage time caused an increase in early-term embryonic deaths (Aygun and Sert, 2013).
        
Neither storage time nor treatment method is a factor in the hatching weight of chicks (P>0.05, Table 6). Toplu et al., (2007) reported that storage time had no statistically significant effect on chick hatching weight. This study is similar to the current one in its conclusion that storage time has no significant effect on chick hatching weight. On the other hand, Shahin and Sedeek (2014) reported that the highest weights were found in the chicks which were administered 14% propolis.
 

 
Microbiological analysis
 
Microbiological analysis of the egg shells found no trace of Escherichia coli or Staphylococcus aureus. These two species of bacteria develop under circumstances of poor hygiene, especially the bodily hygiene of employees. This shows that eggs are collected and stored under hygienic conditions. Furthermore, yeast and mold growth did not vary according to treatment group or storage time (P>0.05) and the total amount of aerobic-mesophilic bacteria was low in the 5% and 10% propolis groups, medium in the 0.1% formalin group and high in water and control groups (P<0.05). A highly significant relation was found between storage time and the total number of aerobic-mesophilic bacteria as shown by the findings that the shells of the eggs stored for 3 weeks contained a higher rate of total aerobic-mesophilic bacteria than those stored for 1 week and 2 weeks (P<0.0001, Table 7). Some researches argued that the best results in the total aerobic-mesophilic bacteria, coliform and Staphylococcus counts on eggshell surface were obtained with propolis and that this safe, non-toxic product was a good alternative to keep microbial load in check in the storage and incubation periods (Shahein and Sedeek, 2014; Aygun and Sert, 2013; Aygun et al., 2012). A separate study, considering the bacteria count on the 18th day of incubation, no significant bacteria growth was reported in the eggs disinfected with chloride, 6% propolis and 9% propolis, however the bacteria growth was found to be at significant levels in the group disinfected with 3% propolis and that it was in fact at pre-disinfection levels. As a result, it was interpreted that the efficacy was not sustainable in the disinfection with 3% propolis (Ýbas, 2018).
 

As the use of high-capacity incubators become more widespread, poultry hatcheries provide eggs from different breeders and send the chicks produced to various enterprises themselves. This requires the development and administration of an effective disinfection procedure to prevent transfer of disease from farm to farm, to maximize the number of chicks hatched, and increase profitability. Substances used in egg disinfection should be effective on microorganisms, but even more important than that they should not have any detrimental or harmful effects on human and animal health. Based on the findings of this study, the use of propolis as a non-toxic substance is proposed for the disinfection of hatching eggs. Propolis has no negative effects on hatchability, it keeps the microbial load in check during storage and can be safely in hatcheries used.