Impact of Hatching Egg Disinfection on Hatching Characteristics and Chick Embryos

The relationship between egg water loss and survival of the embryo may be related to the difference in the ability of embryos to regulate their water contents and eggshell conductance (Hansen et al., 2015). It is apparent that the cuticle may be affected by the application of sanitizers so as to alter embryonic development. The role of using disinfectants on egg cuticle layer as closing or opening the shell pore and consequently affect development or embryonic death was reported by many authors (Copur et al., 2010; Wells, et al., 2011; Girardeau et al., 2014).
 
Several scientists have concluded that the conductance of the eggshell is inversely related to the length of the incubation period for eggs of a known weight (Fasenko et al., 2009). Water loss constant and water vapor gradient formed between the inside of the egg and the external environment can be utilized to generate a direct relationship between water vapor conductance of the eggshell and incubation time noted that male chicks tended to hatch about three hours later than females.
 
The mobilization of calcium causes the eggshell and pore length to thin with some degrees (Iqbal et al., 2016). Also, early maturity birds produce smaller egg weight. The essential oil of oregano has anti-bacterial, anti-oxidant (Rodriguez-Garcia et al.,  2016, cytotoxic (Copur et al., 2010), insecticidal (Traboulsi et al., 2002) and nematicidal (Cumin, the rich oil of the cumin seed contains powerful compounds. These natural constituents possess remarkable antioxidant, antitoxic, anti-microbial, anti-fungal, anti-parasitic, anti-spasmodic and diuretic actions (Elizaquívelet_al2014; Borst, et al., 2014; Rhomerg, et al., 2015; Chen, et al., 2016), formaldehyde is considered to be an irritant to the eyes and the nose, has a lingering noxious odour, carcinogenic effect and venting of  its vapours  is  difficult, different  methods  of shell treatment used during the pre-incubation and  incubation stages are biologically effective on avian embryogenesis (Maharjan, et al., 2017). Colloidal silver  (nanosilver) particles and its derivations have received  attention by some  researchers (Chmielowiec-Korzeniowska et_al2013; Rhomberg, 2015; Maharajan et al., 2017; Al-shammari et_al2015). Fumigation near the time of hatching can also result in embryo mortality (EPA, 2008; Fasenko et al., 2009; Chang et al., 2016) increased fetal defects as  cryptochordism and aberrant (Chang et al., 2015; Rhomerg 2015; Chen, et al., 2016; Maharjan et al.,  2017).

This experiment was carried out to investigate the effect of egg disinfectants from different sources on egg weight loss percentage during incubation, embryonic mortality and hatching characteristics.

Four thousand two hundred eggs from Bandarah chicken strain were used in this experiment. Fresh eggs were collected manually four times daily from the nests. Faecal contaminated eggshells and eggshells with visible cracks were discarded.
 
The eggs were divided into 14 groups, which representing the disinfectants used, as disinfectants from chemical sources and others from natural sources with their combinations, formaldehyde fumigation and control groups. For studying the hatching parameter, each group contains 100 hatching egg and replicated three times for each treatment.
 
Sterile suitable containers were used for dipping the hatching eggs in each disinfectant separately. Eggs were dipped in the disinfectant solutions or fumigated within four hours after collection (Turpin (2013).
        
Untreated control
 
Control with water; control with ethanol alcohol per liter taped water) to make sure that there is any synergistic effect or not, because, oil has been solved in the alcohol. Sodium chloride10%; Hydrogen peroxide5%; Betadine 2%; Virkon S 0.5%; all these chemical disinfectants diluted in 1 L of tap water. While the natural disinfectants: Oregano (0.2.%); Oregano (0.4%); Cumin (02%); Cumin (0.4%); (Oregano 0.1%+Cumin 0.1%); (Oregano 0.2%+Cumin 0.2%) dissolved in 125 ml of Alcohol .
 
Egg was treated for 20 minutes with formaldehyde fumigation (triple strength), 1gm (KMnO₄) to 2ml (CH₂CO) /1m³ (USDA, 1985). Embryonic mortality percentage expressed as a percentage of fertile eggs set was recorded every day of incubation and classified into three periods (1-7, 7-14 and 14-pipping days).
 
At the end of incubation, all hatched eggs were opened and examined macroscopically for evidence of stage for embryonic development. Egg contents were removed and leaving the shell intact. Eggshell thickness without membranes was measured in three middle region areas of each egg for each stage with a micrometer capable of 0.01 mm accuracy and averaged. Hatchability was expressed as percentages of total and fertile eggs. All percentages data of hatchability were subjected to arcsine square root percentages transformation prior to analyses. All percentages data of hatchability were subjected to arcsine square root percentage transformation prior to analyses.   
 
Beginning at 465 hr of incubation and at 6 hr intervals thereafter the hatcher was opened. Chicks that had fully emerged from eggs were removed, wing banded, weighed to the nearest 0.1 gm and recorded as chick body weight at hatch then placed again to the incubator after recording the time of hatch. Hatch time and body weight at hatch were monitored every 6 hours after the hatch of first chick. The chicks were left in the incubator until servicing time (termination of incubation). All chicks were weighed again at the time of removal from the hatcher at 503 hr. Chick weight at pull out is the weight of the chicks at the time of removal from the Hatcher.
 
 Chick body weight loss percentage during incubation and calculated as follows:-  
Chicks for either males or females were reared together and sex was determined at the end of the 6 weeks of growing period. Reversing back to the enumerating eggs and wing-banded chicks, hatching time for both males and females was compared. Also, chick weight at hatch and chick weight at pull were determined for both sexes.
 
The statistical analysis was conducted using SAS program (SAS, 1998) software.

Effects of hatching egg disinfection on embryonic mortality during different stages of incubation are summarized in Table 1. Significantly higher (P<0.05) percentage of embryonic mortality during the early stage of incubation (1-7 days) was observed in egg subjected to formaldehyde fumigation (8.45%), followed by control untreated groups (5.24%) and with water (4.94%) with the same significant difference compared to other treated groups. The lowest significant (P<0.05) percentages of dead embryos at the same first stage of incubation were detected in oregano, cumin and oregano+ cumin with both concentrations compared with the other groups.
 

 
Data obtained concerning the effect of hatching egg disinfectants from different sources and formaldehyde fumigation on embryonic mortality throughout different stages of incubation are shown in Table 2. This table reveals that eggs subjected to formaldehyde fumigation had highest significant (P< 0.05) increase of embryonic mortality percentage at the early stage of incubation through the first seven days (8.45%) and at the mid stage from 7 to 14 days (5.24%).
 

 
Generally, regardless of egg treatment, highest percentages of embryonic mortality had been recorded on days 5 and 6 of incubation. It appears from this figure that each group of disinfection exhibits different patterns of embryonic mortality. Eggs treated by formaldehyde fumigation recorded relatively a higher percentage of embryonic mortality on days, 1, 2, 6 and 17 which being 17, 2, 2 and 2%, respectively. Moreover, natural disinfectant recorded the highest embryonic deaths on days 7 and 17 of incubation being 0.9 and 1%, respectively.
 
Effects of hatching egg disinfection on eggshell thickness (mm) at different stages of embryonic development are recorded in Table 3. The obtained results indicated that the thickest eggshell (0.3 1mm) was recorded for betadine group in infertile eggs. Also, this thickness was significantly (P<0.05) highest compared with those for formaldehyde fumigation and oregano+cumin 0.1%, the highest record of shell thickness was observed for eggs of oregano+cumin 0.1% group(035mm) compared with all other treatment groups with significant differences with those for formaldehyde fumigation and hydrogen peroxide.
 


Eggshell thickness (mm) of various egg types of embryonic development as affected by hatching egg disinfectants from chemical and natural sources compared with formaldehyde fumigation is shown in Table 4. Average of eggshell thickness did not significantly differ among all egg treatments with respect to infertile eggs, early and mid-embryonic mortalities and pipped eggs.
 

 
Generally, results in Tables 1 and 2 revealed that overall mean of embryonic mortality percentage during the first stage of incubation (1-7 day) was higher than those occurred in mid and late stages. Results reported in Tables 1 and 2 are keeping with that reported by other researchers as (Vilchez et al., 1991; Aygun and Sert, 2013; Copur, et al., 2010; Cristina et al., 2015; Hansen, et al., 2015).
 
Several explanations were reported regarding the embryonic mortality during early and late stages of incubation. Eggs with embryos dying late in incubation have decreased in conductance rate (Andersson, et al., 2015; Yamak, et al., 2014). Also, on the other hand, Vilchez et al., 1991, found that increasing embryonic mortality was recorded during the last stage of incubation. This could indicate that optimum air exchange is an important for the early growing as for the high metabolism for the late embryo observed marked improvements in late embryo survival when eggshell permeability was increased.
 
Results of total embryonic mortality for formaldehyde fumigation group was the worst one compared to other natural, chemical and control treatments. It could indicate that formaldehyde fumigation may have a toxic effect on embryos viability.

The results of the present investigation which reveal the increase of embryonic mortality and decrease of hatchability percentage in fumigated egg group and the reverse in oregano and cumin egg groups which demonstrate the increase of hatchability with the decrease embryonic mortality are in accordance with those reported by who mentioned that hatchability percentage in formaldehyde group has been lesser than that of oregano+ cumin (Walls et al., 2011, Rodriguez-Garcia et al., 2016).
 
Results of hydrogen peroxide in Table 4 demonstrated the reduction of embryonic mortality during the experimented stages of incubation compared to those in control are in agreement with those of who reported similar results with using 5% hydrogen peroxide as sanitizing agent Moreover, Beleh (2008) reported that it is worth noting that eggs in control treatment has the highest embryonic mortality but it is normal due to the effect of disinfectants as hydrogen peroxide. Moreover, disinfectant Virkon S was indicated as the worst for early embryonic mortality.
 
Taken together, results of hatchability percentages either for total or fertile eggs and embryonic mortality either total percentage or during different incubation periods for acidic water (hydrogen peroxide and sodium chloride) demonstrated that percentages of these disinfectants recorded better results than those for control groups and worse than those for natural disinfectants. Previous results regarding hydrogen peroxide and sodium chloride compared to control revealed that although treating hatching eggs with acidic water, hatchability did not improve in the egg tested but and it is important to establish that acidic water did not negatively affect embryo health (Fasenko et al., 2009; Ketta and Tùmová 2016). Furthermore, clear change in shell thickness during incubation as was documented for some species. Also, thicker shells produce greater resistance to gaseous diffusion.

As hydrogen peroxide acts as acidic water and affects the shell thickness (Kibala, et al., 2015). Therefore, this disinfectant represents the thinnest eggshell thickness compared with others. This result is consistent with report of Berrang et al., (2000); Iqpal et al., (2016); Leyva-López N.,et_al(2017) which showed that some of the chemicals were noted to affect eggshell characteristics such as porosity and thus are not recommended. 
 
Our results are in accordance with those obtained by who showed that female chicks hatched earlier than males with a difference of about 3 hours between peak hatching time. In addition, the dehydration due to the length of time between hatching and removal of the chick from the hatcher might produce lighter females Moreover, about fifty percent of the female chicks hatched earlier than males (Blanco et al., 2016; Yan et al., 2014).

In conclusion, using natural disinfectants such as oregano 04%, cumin 02% and oregano-cumin 01% could be recommended for realizing the best results of hatch, hatch time, chick body weight and less percentage of chick weight loss and confining hatched chicks in shortest time.