Abstract
Cleanliness of eggs is critical in successful hatching egg operations. The objective of this study was to investigate the effect of trans-cinnamaldehyde nanoemulsion (TCNE) wash treatments, as a sanitation strategy, on embryonic development in fertilized eggs. Trans-cinnamaldehyde is a generally recognized as safe status phytochemical obtained from cinnamon bark. TCNE were prepared with emulsifiers Tween 80 (Tw.80) or gum Arabic and lecithin (GAL) by sonication. Day-old fertilized eggs were subjected to TCNE wash treatments at 34°C for 5 min, followed by 18 d of incubation at 37.7°C. Washing of fertilized eggs with TCNE-Tw.80 or GAL at 0.48% concentration did not significantly alter the egg weight at d 18 of incubation, as compared to baseline and control (P > 0.05). The egg weight loss (calculated as percentage) did not differ significantly between eggs subjected to nanoemulsion wash treatments and control eggs (P > 0.05). In case of embryo fertility and mortality, for baseline and control, ∼ 95% fertility rate was achieved, with combined early and midterm mortality at 16%. Likewise, TCNE-Tw.80 or TCNE-GAL resulted in 95% fertility (P > 0.05), with 11% and 17% combined early and midterm mortality, respectively. Furthermore, TCNE wash treatments did not differ significantly in yolk sac and embryo weight (as compared to control) and did not affect the length of the d 18 embryo (P > 0.05). Moreover, TCNE wash treatments did not alter tibia weight and length (P > 0.05). Results suggest that TCNE could potentially be used as a natural antimicrobial for fertilized egg sanitation. Further studies in industry settings are warranted.
Key words: trans-cinnamaldehyde, nanoemulsion, food-grade emulsifier, embryo development, fertilized egg
INTRODUCTION
Sanitation of hatching eggs is critical in successful hatching egg operations. Recently, chick quality has acquired significant attention by hatcheries and broiler producers as it is an indicator of post hatch broiler performance (Şahan et al., 2014). Therefore, strengthening the efficiency of incubation processes and increasing the quality of day-old chicks are the main aspirations for the broiler industry (Oliveira et al., 2020). Poor sanitation practices can result in early death of embryos, which in turn affects hatchability and chick quality (Shahein and Sedeek et al., 2014). Hatching eggs can become contaminated after being laid by the hen via trans-shell contamination when the egg encounters environmental sources such as feces, litter or water that accommodates pathogenic microorganisms (Upadhyaya et al., 2015). These contaminated eggs disseminate microorganisms in the incubators and hatchers, thereby contaminating other embryonated eggs, and reducing hatchability and chick quality (Shahein and Sedeek et al., 2014; Upadhyaya et al., 2015). Therefore, it is important to implement effective hatching egg sanitation to achieve increased hatchability and to ensure the production of high-quality chicks. In commercial hatcheries, formaldehyde fumigation has long been used to reduce contamination on embryonated eggs. However, potential human toxicity and adverse effects in developing embryos have been reported (Cadirci, 2009). Chemical disinfection using chlorine, quaternary ammonium compounds, hydrogen peroxide, ozone, and electrolyzed oxidizing water have been used (Upadhyaya et al., 2015). However, these strategies have demonstrated limited effectiveness and do not render eggs pathogen-free.
Plant-derived antimicrobials such as trans-cinnamaldehyde (TC), a major component of bark extract of cinnamon (Cinnamomum zeylandicum) have been proven to be an effective antimicrobial for sanitizing table (Upadhyaya et al., 2013) and embryonated eggs (Upadhyaya et al., 2015). The plant-derived antimicrobials are classified by the Food and Drug Administration (FDA) as generally recognized as safe (approval TC-21CFR182.60). However, the poor water solubility of TC is a major challenge for widespread application of the compound in the industry. In our recent publication (Allen et al., 2023), we developed TC nanoemulsions (TCNE) to improve the dispersion and antimicrobial efficacy of TC and demonstrated the efficacy of TCNE in inactivating Salmonella Enteritidis on table eggs. However, there is no data on the safety of TCNE wash treatments on embryonated eggs. Therefore, in this research note, the effect of TCNE wash on the viability and development of embryos in fertilized chicken eggs was studied.
MATERIALS AND METHODS
Preparation and Characterization of Trans-Cinnamaldehyde Nanoemulsion
Trans-cinnamaldehyde nanoemulsions were prepared as described previously (Allen et al., 2023). Briefly, TC oil was dispersed in an aqueous medium to formulate an oil-in-water nanoemulsion using sonication, a high energy method. TC (99%, catalogue no. AC110350010, Fisher Scientific, Waltham, MA) was combined with 2 kinds of emulsifiers, either Tween 80 (Tw.80; catalog no. 28329, Fisher Scientific, Waltham, MA), a synthetic nonionic surfactant or a mixture of gum Arabic (catalogue no. G9752, Sigma-Aldrich, St. Louis, MO) and lecithin (GAL; catalogue no. O3376250, Fisher Scientific, Waltham, MA). The first kind of TCNE prepared with Tw.80 involved homogenizing hydrophobic oil and surfactant at a 2:1 mass ratio for 30 min at constant speed (200–400 rpm). Under continuous stirring, deionized water (DI water) was added dropwise to the oil phase and stirred for 30 min at 23°C. The mixture (5% stock solution) was subjected to continuous sonication for 20 min using an ultrasonicator (QSonica 700, QSonica L.L.C, Newton, CT) at 75 W at 23°C. The second kind of TCNE prepared with GAL was conducted by dissolving gum Arabic and lecithin separately in water at 4% (w/v) to produce stock solutions (Hu et al., 2016). The stock solutions were mixed at equal volume in DI water to prepare aqueous phase. Different concentrations of oil (1.7, 1.25, 0.8, or 0.4%) were first premixed with ethanol and used as a co-surfactant. The mixture was added dropwise to aqueous phase containing DI water and GAL, then mixed for 30 min at 23°C. After mixing oil and aqueous phase, the mixture (1.25% stock solution) was subjected to sonication for 10 min at 75 W, with 10 s pulse On and 5 s pulse Off cycle.
The freshly prepared nanoemulsions were diluted 1:2 with DI water and then subjected to physicochemical characterizations, including particle size, polydispersity index and zeta potential (ζ), determined by dynamic light scattering at 25°C using a Nano Zeta-sizer ZS (Malvern Instruments Ltd, Malvern, WR, UK). The measurements were recorded from 3 replicates of stock solutions.
Effect of TCNE Wash Treatments on Embryonic Development in Fertilized Eggs
Freshly laid hatching eggs from Single-Comb White Leghorn layer chickens were obtained from the University of Connecticut poultry farm. Eggs were transported to Agricultural Biotechnology Laboratory, weighed, then washed in treatment solutions (10 eggs per treatment) consisting of 1,500 mL sterile DI water composed of 0.02% or 200 ppm chlorine, TCNE-Tw.80, GAL or TC 0.48% at 34°C for 5 min. Unwashed eggs or baseline were included in the experiment. Once treated, eggs were kept for drying under a laminar flow hood for 30 min at 23°C. Eggs were transferred to a Hova-Bator operated at 37.7°C, a relative humidity of 58%, with automatic turning every hour at a 45° angle for 18 d of incubation. On the 18th d, eggs were cracked open and evaluated using the following embryonic development parameters (Shahein and Sedeek et al., 2014; Yin et al., 2017; Oliveira et al., 2020). The developmental stage of each embryo was determined according to the Hamburger-Hamilton scale (Hamburger and Hamilton, 1951).
At the end of the incubation process, the percentages of egg weight loss (%), fertility rate (%), early embryo mortality (%), midterm embryo mortality (%), relative embryo weight (%), relative yolk sac weight (%), and tibia weight (%) were calculated as per Oliveira et al., 2020 and Yin et al., 2017 using Equations 1 to 6, respectively. Tibia length and embryo length were measured using a measuring instrument and expressed in centimeter (cm) as indicated in Equations 6 and 7.
-
1.
Egg weight loss (%): [(initial egg weight - egg weight measured after incubation/initial egg weight) × 100]
-
2.
Fertility rate (%): [(number of fertilized eggs/number of eggs set) × 100]
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3.
Early embryo mortality (%): [(number of dead embryos on d 0-6 of incubation/number of fertilized eggs) × 100]
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4.
Midterm embryo mortality (%): [(number of dead embryos on d 7-18 of incubation/number of fertilized eggs) × 100]
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5.
Relative embryo weight (%): [(embryo weight/egg weight) × 100]; Relative yolk sac weight (%): [(yolk sac weight/egg weight) × 100]
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6.
Tibia weight: (%) [(tibia weight/embryo weight) × 100]. Tibia length was measured using a measuring instrument, expressed in cm
-
7.
Embryo length (cm): tip of the beak to the tip of the middle toe
Statistical Analysis
The experiment was conducted using a completely randomized design, consisting of 10 eggs/treatment (N = 60). The study was repeated 2 times. Data were pooled and analyzed using one-way analysis of variance (ANOVA) of R software (version 4.0.2). Differences among means were detected at P < 0.05 using the Tukey's test with appropriate corrections for multiple comparisons.
RESULTS AND DISCUSSION
One of the main strategic points at which the poultry industry can improve production is the sanitation of fertile eggs (Oliveira et al., 2020). Reducing pathogen load on fertile eggs directly translates to improved embryonic development, hatchability, and chick quality (Shahein and Sedeek et al., 2014). Antimicrobial wash treatments must be effective in reducing microbial load on eggshell surface as well as be safe for its application on fertilized eggs in commercial hatcheries. In commercial hatcheries, formaldehyde fumigation of fertile eggs is routinely employed for disinfection, however adverse health effects have been reported in developing embryos and humans in its use (Cadirci, 2009). In our previous publication, we investigated the antimicrobial efficacy of TCNE on table eggs and observed that TCNE wash was very effective in reducing Salmonella load (Allen et al., 2023). Therefore, in this study, we evaluated the effect of TCNE on embryonic development at 37.7°C for 18 d of incubation (Shahein and Sedeek et al., 2014; Yin et al., 2017; Oliveira et al., 2020). We selected 18 d as our endpoint for this study because in a commercial setting, eggs are transferred at d 18 to hatchers with temperature and humidity requirements of 36.9°C and 66 to 75% respectively. Therefore, our primary goal for this project was to observe the effect of TCNE wash treatments on embryo development in the first 18 d. Chlorine wash was included as an industry control because chlorine is also used for hatching egg sanitation. We selected 2 kinds of emulsifiers for nanoemulsion preparation, Tw.80 and GAL, which are food-grade and approved by FDA as generally recognized as safe for applications in foods (Tw.80 approval: 21CFR172.840; GAL approval: 21CFR184.1330 and 21CFR184.1400). Their main function is to decrease the size of oil droplets and minimize the interfacial tension between the aqueous and oil phase to construct nanoscale oil droplets (Hu et al., 2016). We observed that TCNE prepared with Tw.80 or GAL were within acceptable range of particle size, polydispersity index and zeta potential of being classified a nanoemulsion of high quality when stored at room and refrigeration temperatures for 8 wk (Allen et al., 2023).
The effect of TCNE treatments on egg weight after 18 d of incubation is depicted in Table 1. Washing fertilized eggs with water (control) or water containing chlorine 200 ppm, TCNE-Tw.80, TCNE-GAL or TC 0.48% for 5 min did not significantly alter the egg weight at d 18 of incubation, as compared to baseline or unwashed eggs (P > 0.05). As embryos develop within the eggshell, the egg naturally loses weight. The sanitizer used for disinfection may affect egg weight, as the cuticle layers and permeability of shell may be altered. The egg weight loss (calculated as percentage) did not differ significantly between treatments, control, and baseline (P > 0.05). In a separate study, Oliveira et al. (2020) observed a similar result where clove essential oil treated fertilized eggs did not differ significantly in egg weight loss percentage between treatments. The effect of TCNE treatments on fertility, early and midterm embryo mortality, relative yolk sac weight, embryo weight and length, and tibia weight and length are presented in Table 2. In baseline and control eggs, ∼ 95% fertility rate with overall mortality of 16% (mortality %: number of deceased embryos/number of fertilized eggs × 100) was observed. Likewise, TCNE-Tw.80 or TCNE-GAL resulted in 95% fertility (P > 0.05), with 11% and 17% combined early and midterm mortality, respectively. All the eggs subjected to chlorine, TCNE-Tw.80, TCNE-GAL or TC 0.48% wash treatments did not differ significantly in fertility and combined early, and midterm mortality percentage as compared to baseline and control (P > 0.05). In parallel with Oliveira et al. (2020), no significant difference in fertility and mortality percentages during the early or medial stage of incubation between the treatments was observed.
Table 1.
Effect of trans-cinnamaldehyde nanoemulsion (TCNE) on egg weight prior to setting and 18 d post incubation at 37.7°C and the percentage of egg weight loss in eggs treated with different treatments.1
| Treatments | Initial egg weight (g) | Final egg weight (g) | Egg weight loss (%) |
|---|---|---|---|
| Baseline | 63.78 ± 0.64a | 58.98 ± 0.66a | 7.45 ± 0.94a |
| Control | 62.91 ± 0.94a | 58.17 ± 0.91a | 7.46 ± 0.95a |
| Chlorine 0.02% | 63.48 ± 0.90a | 58.50 ± 0.87a | 7.75 ± 1.02a |
| TCNE-Tw.80 0.48% | 63.33 ± 0.74a | 58.53 ± 0.74a | 7.52 ± 0.89a |
| TCNE-GAL 0.48% | 62.52 ± 0.86a | 57.71 ± 0.86a | 7.63 ± 0.99a |
| TC oil 0.48% | 62.73 ± 0.84a | 57.89 ± 0.85a | 7.67 ± 0.80a |
Data presented as mean ± standard error of mean. The treatments included baseline (unwashed egg), control, chlorine (0.02%), TCNE- Tw.80, GAL or TC (0.48%) at 5 min of treatment. Each experiment consisted of 10 eggs per treatment and repeated at least twice.
Values within column with same superscript did not differ significantly (P > 0.05).
Table 2.
Effect of trans-cinnamaldehyde nanoemulsion (TCNE) on fertility, early and midterm embryo mortality, relative yolk sac weight, embryo weight and length, and tibia weight and length according to different treatments.1
| Treatments | Fertility rate (%) | Early mortality (%) | Midterm mortality (%) | Yolk sac weight (%) | Embryo weight (%) | Embryo length (cm) | Tibia weight (%) | Tibia length (cm) |
|---|---|---|---|---|---|---|---|---|
| Baseline | 95 ± 5.00a | 5 ± 5.00a | 11 ± 11.11a | 20.10 ± 1.08a | 37.30 ± 1.59a | 12.52 ± 0.20a | 0.28 ± 0.02a | 1.98 ± 0.07a |
| Control | 95 ± 5.00a | 11 ± 0.50a | 5 ± 5.00a | 19.27 ± 0.99a | 35.78 ± 2.45a | 12.06 ± 0.44a | 0.29 ± 0.04a | 1.94 ± 0.12a |
| Chlorine 0.02% | 95 ± 5.00a | 5 ± 5.00a | 5 ± 5.00a | 19.06 ± 1.38a | 34.82 ± 1.96a | 12.34 ± 0.33a | 0.26 ± 0.04a | 1.77 ± 0.09a |
| TCNE-Tw.80 0.48% | 95 ± 5.00a | 5 ± 5.00a | 6 ± 5.50a | 20.71 ± 0.79a | 36.95 ± 2.00a | 12.22 ± 0.38a | 0.30 ± 0.05a | 1.93 ± 0.09a |
| TCNE-GAL 0.48% | 95 ± 5.00a | 6 ± 5.50a | 11 ± 0.50a | 20.85 ± 1.07a | 38.53 ± 2.04a | 12.43 ± 0.38a | 0.30 ± 0.04a | 2.03 ± 0.09a |
| TC oil 0.48% | 100 ± 0.00a | 0 ± 0.00a | 5 ± 5.00a | 19.94 ± 1.03a | 35.11 ± 2.46a | 11.72 ± 0.43a | 0.31 ± 0.05a | 1.89 ± 0.09a |
Data presented as mean ± standard error of mean. The treatments included Baseline (unwashed egg), control, chlorine (0.02%), TCNE-Tw.80, GAL or TC (0.48%) at 5 min of treatment. Each experiment consisted of 10 eggs per treatment and repeated at least twice.
Values within column with same superscript did not differ significantly (P > 0.05).
For hatcheries and broiler producers, chick quality is crucial in determining post hatch broiler performance (Şahan et al., 2014). Therefore, in the present study, we measured embryo weight and length, yolk sac weight as well as tibia weight and length to evaluate embryo quality. We observed no significant difference in yolk sac weight, embryo weight and embryo length between treatments, and control, after 18 d of incubation (P > 0.05). Moreover, nanoemulsions did not affect tibia weight and length (P > 0.05). To our knowledge, this is the first study that has explored the effect of plant-based nanoemulsions on embryo development in a fertilized egg. In conclusion, TCNE prepared with Tw.80 or GAL had no deleterious effects on embryonic development, validating its safety for application on fertilized eggs. However, future investigations testing the effects of TCNE wash on fertilized egg hatchability are warranted in an industry setting.
ACKNOWLEDGMENTS
This research was supported in part by USDA-ARS-ATA Research and Small and Medium Sized Farms program grant (#2020-69006-31684; Accession # 1022281) from the USDA National Institute of Food and Agriculture.
DISCLOSURES
The authors declare no conflicts of interest.
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