Essential oil sanitizers to sanitize hatching eggs

ABSTRACT

Background: Eggs are low-cost source of protein for balanced human diet due to the high contents of essential nutrients. On eggshells, various microbial groups exist that may affect egg quality, embryonic development, and human health, posing potential health, food safety, and economic concerns. The use of essential oils to decontaminate eggshells could be a effective strategy for eliminating pathogenic microbes. Thus, the current study was designed to investigate the efficacy of oregano and cumin essential oils in controlling microbial activity on eggshell and assessing their effects on fertile egg hatchability, embryonic growth, and chick development after hatching. Methods: Hatching eggs were divided into four groups; control groups, fumigation, treatment with Oregano oils 0.5% and treatment with Cumin oils 0.5%. The bacterial contaminants of eggshell were enumerated for each experimented groups. The effects of the hatching egg disinfection on growth parameters including fertile egg weight, egg weight loss percentage, macroscopic fertility and hatchability percentages were estimated. Results: Total bacterial counts were significantly reduced in all treated experiments compared with those of the control ones. Microcephaly and exencephaly were clearly observed in the embryos treated with formalin fumigation. All embryos treated with oils exhibited no observed malformation on brain and spinal cord.

KEYWORDS:

• Bacteria
• cumin
• decontamination
• eggshells
• Oregano

1. Introduction

The Size of poultry hatchery operations has grown over time. This change has raised hopes for improved hatchability, lower chick mortality and improved bird performance. Until recently, knowledge of incubation was not as advanced as knowledge of other aspects of the chicken production chain. The knowledge gained over the last few years in poultry nutrition, health, management and the environment has not been transferred to the field of incubation. Incubation factors were only recently recognized as significant influences on chicken performance. An effective hatchery sanitation programme is essential for achieving a high level of hatchability and producing high-quality chicks. (Park and Sohn 2018).

Because of their high protein, fat and other essential nutrient content, eggs are an essential part of the human diet. On eggshells, various microbial groups exist, including Enterococcus spp. (Lanza et al. 2022), Salmonella spp. (Haubert et al. 2022) and Turicibacter bilis (Maki and Looft 2022). Pathogenic bacterial strains have also been found on eggshells. When contaminated food is consumed, Salmonella enterica subsp. enterica serovar Enteritidis. The abbreviation is Salmonella Enteritidis., a bacterial pathogenic agent of poultry, that can cause foodborne illness in humans such as gastroenteritis. Such microbiota are more likely to be found in hens’ cloaca and feathers. Microbes on eggshells may have an impact on embryonic development, resulting in significant economic losses. The ventilation system easily spreads egg microbial populations through the hatcher and then cycles them into setters and hatchers. The presence of foodborne pathogens on eggs raises health and food safety concerns.

Eggshell decontamination is regarded as an effective strategy for eliminating pathogenic microbes and making eggs safe for human consumption. Several techniques for sanitizing eggshells have been suggested, including chemical and radiation treatments (Chousalkar et al. 2021). However, implementing these approaches has serious consequences for egg quality, human health and the environment. As a result, finding a safe alternative method, such as using essential oils to decontaminate eggshells, is of interest.

Essential oils are major secondary metabolic products obtained from various plant organs such as roots, stems, leaves and fruits (Oliveira et al. 2020). The essential oil of oregano (Origanum vulgare L.) and Cumin (Cuminum cyminum L.) have exhibited antimicrobial activities (Karik et al. 2021 and Xu et al. 2021).

The essential oil of oregano has anti-bacterial properties mainly due to the basic constituents including β-pinene (0.5%) – thymol (0.86%), terpinene and p-cymene – Carvacrol (57.01%). Human health, environmental concern and consumers’ demand for residue-free food necessitate the evaluation of alternative, reduced-risk control methods. (Vági et al., 2005).

Cumin, the rich oil of the cumin seed contains powerful compounds. The predominant compound, cuminaldehyde, accounts for up to 40% of the oil content. Aldehydes are rich in naturally occurring oxygen compounds which can interact with human cells to make profound beneficial changes. These natural constituents possess remarkable antioxidant, antitoxic, antimicrobial, antifungal, anti-parasitic, anti-spasmodic and diuretic actions (Tepe et al., 2005).

Formaldehyde (CH2CO, formalin, formol) is a gas at room temperature and it is readily soluble in water. It is commonly used as a disinfectant, as it is cheap, not corrosive and kills most bacteria and fungi (including their spores). The biocidal efficacy of formaldehyde is due to its ability to act on the proteine and nucleic acid bases of microorganisms (In addition, formaldehyde also alkylates the nitrogen atoms of purine and pirimidine bases in DNA and RNA) (Chousalkar et al. 2021). It has demonstrated the importance of the hatched chick’s early nutritional status in boosting performance. It is crucial that when developing diets for parent birds, consideration is given not only to maximising egg output and hatchability, but also to ensuring that the eggs produced contain adequate amounts of the nutrients required to support the early development of the hatching chick.

Therefore, the aim of the current study was to investigate the effectiveness of Oregano and Cumin essential oils, to control microbial activity on eggshell and to assess their impacts on hatchability of fertile egg, embryonic growth and development of chicks post-hatching.

2. Material and methods

The present experiment studies were conducted at the Poultry Research Station and Biological Sciences Department, College of Science, King Faisal University during the period from November 2021 up to February 2022. All experimental procedures will be done according to research ethics (Reference number: KFU-REC-2022-FEB-EA000436).

2.1. Collection of hatching eggs

A total of 540 hatching eggs of local chicken strain was used in this experiment. Hatching eggs were divided into three divisions: 140 hatching eggs for bacterial count, secondly, 200 hatching eggs were used for hatching parameters and thirdly, 200 eggs for embryonic inspection. Fresh eggs were collected manually four times daily from the nests. Faecal-contaminated eggshells and eggshells with visible cracks were discarded.

The eggs for each division were divided into four groups, which are: control groups (GI) (untreated, and treated with alcohol), formaldehyde fumigation (GII), (GIII) treatment with Oregano oils (5%) 0.55 μl/cm3and (GIV) treatment with Cumin oils (5%) 0.55 μl/cm³.

The basis of drug dose selection in our study was our own previous study (Zeweil et al. 2015).

Eggs were dipped in the disinfectant solutions or fumigated within four hours after collection. The disinfectant solutions were used for dipping the eggs for three minutes at 38–40 Co.

Each group for studying the hatching parameter contained 25 hatching eggs and replicated twice for each treatment. Hatching eggs were given formaldehyde preincubation fumigation for 15 min with the recommended concentration of 1.4 ml of formalin added to 0.5 g of potassium permanganate per cu ft. Triple strength formaldehyde gas was produced inside the setter for 20 min (USDA 1985).

Experimental Divisions:

The experiments were divided into three divisions.

Bacterial count on eggshell surface

Incubation and its parameters

Embryogenesis

1. Bacterial count on eggshell surface:

The bacterial contaminants of eggshell were enumerated for each experimented groups using sterile swab on area of 2 cm² for each egg per treatment using sterile flexible template, according the method of Şimşek et al. (2007). The eggs were sampled three times, the first one before treatment, the second one after 60 min of treatment and the third one at the time of setting in the incubator for studying the total bacterial count.

The current study aimed to investigate the effectiveness of Oregano and Cumin essential oils in controlling microbial activity on eggshell and assessing their effects on hatchability of fertile eggs, embryonic growth, and post-hatching chick development.

1. Estimation of hatching eggs’ parameters during incubation

The eggs were serially numbered and weighed before being placed in an incubator set to 37.5°C. and 55% relative humidity. The capacity of the incubator is 3000 hatching eggs and 1000 for the hatcher. The time of egg setting in the setter was recorded for each trial to obtain the exact hatch time in hours and was considered the experiment’s zero hours. To reduce the possibility of position effects, all eggs were set and distributed randomly in different locations on the same trolley of the setter. The eggs were transferred singly into pedigree hatching nests on the eighteenth day of incubation and then placed in a hatcher for the remainder of the incubation period at 37.2°C and 65% relative humidity for 3 days until the hatch.

Throughout the incubation period, the effects of hatching egg disinfection on growth parameters such as fertile egg weight, egg weight loss percentage, macroscopic fertility and hatchability percentages were estimated. The experiment design is ambiguous

All eggs were weighed individually again during incubation on the 5th, 10th, 15th and 18th days and at the first signs of pipping to obtain egg weight loss percentages for each incubation time and calculated as follows according to Bhale et al.(2003): $\begin{array}{rl}& \mathrm{E}\mathrm{g}\mathrm{g}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{s}\text{\%}\\ & ={\displaystyle \frac{\mathrm{E}\mathrm{g}\mathrm{g}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{a}\mathrm{r}\mathrm{l}\mathrm{i}\mathrm{e}\mathrm{r}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{o}\mathrm{d}\text{-}\mathrm{e}\mathrm{g}\mathrm{g}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{a}\mathrm{t}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{o}\mathrm{d}}{\mathrm{E}\mathrm{g}\mathrm{g}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{a}\mathrm{r}\mathrm{l}\mathrm{i}\mathrm{e}\mathrm{r}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{o}\mathrm{d}.}}\\ & \times 100\end{array}$

Hatchability:-

Hatchability percentage was estimated as follows:- $\begin{array}{rl}& \mathrm{H}\mathrm{a}\mathrm{t}\mathrm{c}\mathrm{h}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{e}\mathrm{g}\mathrm{g}\mathrm{s}\mathrm{e}\mathrm{t}\text{\%}\\ & ={\displaystyle \frac{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{h}\mathrm{a}\mathrm{t}\mathrm{c}\mathrm{h}\mathrm{e}\mathrm{d}\mathrm{s}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{c}\mathrm{h}\mathrm{i}\mathrm{c}\mathrm{k}\mathrm{s}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{e}\mathrm{g}\mathrm{g}\mathrm{s}\mathrm{e}\mathrm{t}}\times 100}\end{array}$ $\mathrm{H}\mathrm{a}\mathrm{t}\mathrm{c}\mathrm{h}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{o}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{l}\mathrm{e}\mathrm{e}\mathrm{g}\mathrm{g}\mathrm{s}\mathrm{e}\mathrm{t}\text{\%}={\displaystyle \frac{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{h}\mathrm{a}\mathrm{t}\mathrm{c}\mathrm{h}\mathrm{e}\mathrm{d}\mathrm{s}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{c}\mathrm{h}\mathrm{i}\mathrm{c}\mathrm{k}\mathrm{s}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{l}\mathrm{e}\mathrm{e}\mathrm{g}\mathrm{g}}\times 100}$

All percentages data of hatchability were subjected to arcsine square root percentage transformation prior to analyses.

Embryogenesis:

On all incubation days, each egg was weighed and opened, and the embryos were separated from the remaining egg contents. Three developing embryos were examined for morphological and histological features on each of the preceding days of incubation for each experimental and control group. For histological preparation and studying, embryos were rinsed in saline water and fixed in Bouin^,s fluid for 24 h. Fixation consists of the following compositions: saturated aqueous solution of picric acid 100 parts, formaldehyde solution 25% parts and five parts of glacial acetic acid were added promptly before using. This solution acts as a fixative and/or preservative. After fixation, embryos were thoroughly washed with 70% ethyl alcohol. Then they were dehydrated through an ascending series of alcohol then cleared in xylene and embedded in paraffin. Paraffin blocks were treated, fixed over the block holder of the microtome and serially sectioned at 8–10 μ. The obtained paraffin ribbons containing the serial sections were cut into pieces of 5 cm long and mounted over a slide placed over a hotplate adjusted at 40°C. The mounted sections were allowed to dry completely in an oven adjusted at 40°C for one week. After complete dryness and sticking of the sections over the slides, it was taken out of the incubator and subjected to the staining process, then dehydrated, cleared, mounted in balsam and glass slip

Statistical Analyses:

The statistical analysis was conducted using SAS program (SAS 1998) software, the following model was used: $Y_{ij}=M+L_i+e_{ij}$

where

• Y_ij = observation record,

• M = the overall mean,

• L_i = is the effect of disinfection, i = 1–14 and

• e_ij = is the random error

Means differences were separated by Duncan New Multiple range test (Duncan’s 1955).

3. Results

Data obtained concerning effect of hatching egg disinfection on total bacterial count of eggshell surface are shown in Table 1. Results demonstrated in this table reveal that total bacterial counts on hatching eggshell surface were significantly (P < 0.05) reduced as a result of using all disinfectants compared with those of the control ones. The total bacterial counts decreased from 8.08 log in GI to 3.7, 1.6 and 1.9 log in GII, GIII and GIV, respectively. Similarly, a significant reduction on bacterial count was observed for all groups (after treatments and before setting the eggs in the incubator) except for formaldehyde fumigation group. Bacterial counts in GII were significantly increased from 3.7 log after treatments to 4.7 log before setting the eggs in the incubator.

Table 1. Effect disinfection on total bacterial eggshell count (cfu per egg) (log ± SE) of 2 cm².

Treatment	Total bacterial count
	Before treatment	After treatment	Before setting in the incubator	Significant
Control untreated	8.07 ± 1.34	6.83 ± 1.01^a	7.33 ± 0.88^a	^N.S
Control with alcohol	8.08 ± 1.34^A	3.08 ± 0.18^bB	2.74 ± 0.02^cB	*
Formaldehyde fumigation	8.08 ± 1.34^A	3.73 ± 0.37^bB	4.30 ± 0.40^bAB	*
Oregano 0.5%	8.08 ± 1.34^A	1.60 ± 0.01^bB	1.00 ± 0.68^cB	*
Cumin 0.5%	8.08 ± 1.34^A	1.93 ± 0.18^bB	1.20 ± 0.67^cB	*
Significant	^N.S	*	*

Notes: a, b and c Means within each column for each item with different superscripts are significantly different (P < 0.05).

A and B Means within each row for each item with different superscripts are significantly different (P < 0.05).

N.S: non significant *significant (P < 0.05).

Treatment of the eggs with natural disinfectant Cumin 0.5%, significantly (P < 0.05) reduced the total bacterial count from 8.08 to 1.20 log with 85.6% of reduction before setting in the incubator. Also, treatment of the eggs with natural disinfectant Oregano 0.5%, significantly (P < 0.05) reduced the total bacterial count from 8.08 to 1.00 log with 7.33% reduction before setting in the incubator. Whereas, treatment with formaldehyde fumigation significantly (P < 0.05) reduced the bacterial count from 8.08 to 3.73 log with 46.16% reduction, but the count has increased numerically again during storage to be 4.3 log before setting in the incubator. No significant differences were noticed in the total bacterial counts on all egg groups (GIII and GIV), of treatments with Oregano and Cumin oils.

Data obtained concerning effects of different hatching egg natural disinfectants and formaldehyde fumigation on egg weight during incubation are shown in Table 2. No significant difference in initial egg weight was observed comparable with that on days 5, 10, 15, 18 of incubation and at pipping, among the disinfections egg groups. Highest egg weight was observed on day 5 of incubation onward and at pipping for groups treated with Cumin and Oregano oils. Formalin fumigation resulted in the lowest egg weight in all the incubation intervals (51–43 gm).

Table 2. Effect disinfection on fertile egg weight during incubation.

Treatment	Egg weight through incubation (gm)
	Zero day	5thday	10th day	15th day	18th day	At pipping
Control untreated	52.09 ± 0.24	50.24 ± 0.24^abc	48.68 ± 0.24^abc	47.02 ± 0.24^bcd	45.75 ± 0.23^de	45.22 ± 0.24^cde
Control with alcohol	52.84 ± 0.24	50.72 ± 0.23^c	47.78 ± 0.24^f	46.32 ± 0.24^ef	45.80 ± 0.24^de	45.62 ± 0.24^efg
Formaldehyde fumigation	51.76 ± 0.26	49.22 ± 0.25^ab	47.08 ± 0.25^def	46.02 ± 0.24^bcd	45.22 ± 0.24^cd	43.56 ± 0.24^cd
Cumin 0.5%	51.98 ± 0.23	50.29 ± 0.23^ab	49.16 ± 0.23^ab	47.44 ± 0.24^b	46.44 ± 0.24^bc	46.32 ± 0.25^ab
Oregano 0 .5%	51.92 ± 0.26	50.52 ± 0.25^a	49.55 ± 0.26^a	47.83 ± 0.26^a	46.76 ± 0.26^ab	46.65 ± 0.26^ab
Significant	N.S	*	*	*	*	*

Notes: a, b, c, d, e, f and g Means within each column for each item with different superscripts are significantly different (P < 0.05).

N.S: Non Significant * Significant (P < 0.05).

Table 3 showed the effects of different egg disinfectants and formaldehyde fumigation on egg weight loss percentage throughout incubation. The percentage of egg weight loss from 0 to 18 days ranged from 9.22 to 12.30%. The percentage of egg weight loss through the setting phase of incubation (0–18 days) was significantly increased for the control eggs (∼12.10%) compared with that of GII (10.3%) GIII (9.5%) and GIV (9.2%).

Table 3. Effect disinfection on egg weight loss percentage for eggs.

Treatment	Egg weight loss parentage
	0–18 d	0- Pipping day
Control untreated	12.32 ± 0.11^a	12.78 ± 0.12^ab
Control with alcohol	11.36 ± 0.07^c	12.24 ± 0.07^cd
Formaldehyde fumigation	10.33 ± 0.08^d	11.01 ± 0.06^fg
Cumin 0.5%	9.54 ± 0.11^e	10.88 ± 0.13^fg
Oregano 0.5%	9.22 ± 0.14^e	10.66 ± 0.15^g
Significant	*	*

Notes: a, b, c, d, e, f and g Means within each column for each item with different superscripts are significantly different (P < 0.05).

*significant (P < 0.05).

Table 4 displayed the effects of natural egg disinfectants and egg formaldehyde fumigation on percentages of macroscopic egg fertility and hatchability of fertile and total eggs set. The obtained results indicated that the highest significant (P < 0.05) records of macroscopic egg fertility were observed for groups treated with oregano (97.52%) and cumin (96.72%) compared with those for egg controls (96%). Whilst the lowest percentage of egg fertility was recorded for eggs treated with formaldehyde fumigation (94.64%).

Table 4. Effect of hatching disinfection on macroscopic fertility and hatchability percentages (X ± SE).

Treatment	Macroscopic egg Fertility%	Hatchability of fertile egg%	Hatchability of total egg %
Control untreated	95.22 + 0.22^abc	84.06 ± 1.54^d	82.00 ± 1.52^d
Alcohol control	96.14 ± 0.64^ab	88.66 ± 1.54^cd	87.64 ± 1.45^cd
Formaldehyde fumigation	94.64 ± 0.22^bc	82.05 ± 0.56^f	44.64 ± 0.22^e
Oregano 0.5%	97.52 ± 0.64^a	96.21 ± 05.64^ab	92.96 ± 0.64^a
Cumin 0.5%	96.72 ± 0.22^abc	95.76 ± 0.94^a	91.94 ± 1.20^ab
Significant	*	**	**

Notes: a, b, c, d, e and f Means within each column for each item with different superscripts are significantly different (P < 0.05).

* significant (P < 0.05) **significant (P < 0.0).

Histological examination of the brain in the treated developing embryos is illustrated in Figure 1. Unlike the control (Figure 1(a)), morphological abnormalities were clearly observed in the developmental brain of embryos treated with formalin fumigation (Figure 1(b–d)). Microcephaly, degenerated and obliterated brain with convolutions within the ventricles and non-closed hind brain were observed (Figure 1(b)). Furthermore, microcephaly and microophthalamic (E) embryo with degenerated brain in collapsed cavities and absence of constrictions in brain segments, non-closure of the fore brain and hypo-expanded brain at the FB and MB were noticed (Figure 1(c)).

Figure 1. a. A photograph showing control group with normal structure of the brain chambers, fore brain (FB) mid brain (MB) and hind brain (HB) with clear ventricles. b–d (treated egg with formalin fumigation showing malformed and congenital head and brain), b. microcephaly with degenerated brain (B), the non-closure of the hind brain (arrow). C. Microcephaly and microophthalamic (E) embryo with Malformed and degenerated brain in collapsed cavities, absence of limitation between brain segments and non-closure of the fore brain (arrow), d. Exencephalic embryo and severe reduction of ventricles. Note non-closure of the fore and mid brain (arrows). e. Normal developed brain in the treated case with either oregano or cumin oils.

Exencephalic embryo with severe degeneration of the brain with, demarcated limits between brain chambers and severe reduction of ventricles and eye size, non-closure of the fore and midbrain were also noted (Figure 1(d)). All treated embryos with either Oregano or Cumin oils or control were similar to each other (Figure 1(e,a)), and no evident malformations or differences between them were observed.

Histological examination of the spinal cord in the treated embryos is illustrated in Figure 2. Malformed spinal cord including severe degeneration and haemorrhage in the central canal was reported in embryos treated with formalin fumigation Figure 2(b). Additionally, the spinal cord architecture was dramatically changed into zig zag-like structure with ruptured layers. No marked malformations were observed in treated embryos with either Oregano or Cumin oils or untreated ones (Figure 2(d and a)).

Figure 2. A photograph showing normal structure of spinal cord in the control case with normal architecture of the layers central canal (CC), grey matter (GM) and white matter (WM). b–c showed malformed spinal cord in the eggs treated with formalin fumigation, b. severe degeneration of spinal cord layers (red square) and haemorrhage in the central canal (arrows) resulted in closure of central canal, c. zig zag spinal cord and degeneration of grey or white matter (arrows), d. egg treated with oregano or cumin oils disinfectants showing normal structure of spinal cord layers.

4. Discussion

Eggs Contain high levels of essential nutrients required for balanced human diet. However, pathogenic bacteria-associated eggshells have negative impacts on egg quality, embryonic development and human health posing potential health, food safety and economic concerns (Songsamoe et al. 2022).

There are many factors contributing to the failure of a fertile egg to hatch which include lethal genes, insufficient nutrients in the egg and exposure to conditions that do not meet the needs of the developing embryo. Breeder factors that affect hatchability include strain, health, nutrition and age of the flock, egg size, weight and quality, egg storage duration, sanitization and conditions (Kingori 2011).

Plant’s essential oils particularly oregano (Origanum vulgare L.) and Cumin (Cuminum cyminum L.) could be a promising and safe approach to overcome egg’s infection with harmful microbes. This study aimed to assess the potential antimicrobial activities of Oregano and Cumin essential oils and on hatchability of fertile egg and post-hatching traits.

The finding that the significant decrease in total bacterial count on eggs treated with Cumin or Oregano can be explained by their antimicrobial activities. These results coincide with those obtained by Karik et al. (2021), Oliveira et al (2021) and Xu et al. (2021), who reported that Oregano and Cumin essential oil eliminates microbial populations naturally occurring on the egg shell surfaces. However, the bacterial count increased on eggshells again before setting in the incubator when treated with formaldehyde fumigation could be attributed to the no residual effects of formaldehyde through the storage period. Our results are in agreement with those that showed formaldehyde fumigation is teratogenic and carcinogenic (Bekhet 2021; Li et al. 2021; Oliveira et al . 2021; Kou et al. 2022).

The observation that the egg weight and egg weight loss percentage decreased in GII (Tables 2 and 3) is likely to be due to the penetration of loads of bacterial species from the eggshell to the embryo, consuming internal egg contents. Additionally, fumigation adversely affects the integrity and permeability of egg cuticle leading to excessive water vapour loss (Oliveira et al. 2021). Whilst the increase in egg weight on day 5 of incubation onward and at pipping for groups treated with Cumin and Oregano oils might be related to less bacteria being able to penetrate eggs, indicating no damaging effect on the egg cuticle (Copur et al. 2010; Badran et al., 2018). Comparable results were obtained by Taşdemir et al. (2021) who investigated the effects of oregano juice on eggshell microbial load, layer embryo development, hatching results, and growth pre- and post-hatching.

Moreover results in Table 4 revealed that the highest significant (P < 0.01) percentages for hatchability of fertile egg were recorded in egg groups with oregano 0.5% (96.21%) and cumin 0.5% (95.76%) compared with other disinfected egg groups, formaldehyde fumigated and control groups. Also, highest significant (P < 0.01) percentages for hatchability of total eggs were recorded for groups treated with natural disinfectants with oregano 0.5% (92.69%), cumin 0.5% (91.94%) The higher hatchability rates for eggs sanitized with oregano and cumin oils indicate their friendly effect on permeability of egg cuticle and developing embryo and adversely-effect on bacterial contamination on eggshells. On contrary, formaldehyde fumigation seemed to be toxic for the embryo and accordingly decrease the hatchability.

The normal brain has three discriminated chambers; forebrain, elevated midbrain and hindbrain (Figures 1(a,e)). Formalin fumigation induced severe malformation in the developing brain (Figure 1(b–d)) due to it has severe oxidative stresses on brain and induces misfolded neurones (Rana et al. 2021).

All embryos treated with Oregano and Cumin oils exhibited no observed malformation on brain and spinal cord. Exencephalic embryo with severe degeneration of the brain with demarcated limits between brain chambers and reduction or absence of ventricles also note non-closure of the fore and midbrain and brain tissue remains exposed to the outside. In addition to these CNS malformations, eye size reduction and other general malformations, such as developmental delay and reduced size, were observed.

5. Conclusions

Collectively, Oregano and Cumin oils act as effective disinfectant against microbes on the eggshell by significantly diminishing the bacterial count, increased egg weight, decreased weight loss percentage, increased hatchability. Based on the results described herein, it was recommended that disinfection immediately after laying is strongly advised to avoid any microbial challenge that exist in the hatchery environment. Optimizing poultry production efficiency requires maximizing hatchability and chick quality. reduced microbial contamination of eggshells may aid in the reduction of bacterial infections in developing embryos and newly hatched chicks. These findings open the door for developing natural preservatives to be used in improving egg quality, food safety and industrial poultry production at a commercial scale, in future.

Author contributions

GB: Rearing eggs in the experiment, numerical data analysis and histopathological work, AK: Numerical data analysis and histopathological work. Both authors shared preparing the manuscript.

Acknowledgements

The authors thank the Deanship of Scientific Re-search, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Al-Ahsa, Saudi Arabia, for financial support, [GRANT1,481].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Al-Ahsa, Saudi Arabia [GRANT1,481].