Characterization of zinc oxide nanoparticles (ZnONPs)
The FT-IR spectra of chemically synthesized ZnONPs in the range of 4000-400 cm-1
(Fig 1a). The observed peaks of the FT-IR spectrum depends on the size and morphology of ZnONP were referred from previous works of literature in order to confirm the findings.
Fig 1: Characterization of ZnO nanoparticle (a) FTIR (b) scanning electron microscopy.
The prepared ZnONPs showed infra-red (IR) peaks at 436 cm-1
, 1384.25 cm-1
and 3447.98 cm-1
. The peak positioned at 436.59 cm-1
is attributed to the Zn-O stretching bonds. Ismail et al., (2018)
revealed that IR broad absorption feature positioned at 443.96 cm-1
confirms the stretching vibration of Zn-O.
The absorption peak of hydroxyl group of zinc hydroxide is at 1384 cm-1
, in the spectrum was recorded due to C=O stretching. The adsorbed free CO2
from the air might be responsible for the band at 1384 cm-1 (Nejati et al. 2007)
. The bands in the region of 1700-3300 cm-1
were lost due to the removal of water molecules.
The SEM image in Fig 1b clearly depicted ZnONPs size ranging from 45 to 98 nm. Nearly rod shaped nanoparticles was recorded with an average size of 68 nm. These results were consistent with the previously reported study of Kumar et al., (2022).
Hatchability in negative control group was recorded 73.3% whereas 66.7% was observed in normal saline injected group (T1
). Decreased hatchability in positive control group may be due to the injection process. In ovo
administration of ZnONPs 5 ppm and Vitamin C had around 72% and 76.7% hatchability respectively (Fig 2a). Palouj et al., (2021)
reported reduces hatchability and increased embryonic mortality with in ovo
injection of ZnONP.
Fig 2: Effect of in ovo administration (18DOI) of normal saline, ZnONP and vitamin Con (a): Hatchability percentage; (b): Egg weight and hatch weight of chicks; (c): Chick weight to egg weight ratio, of day old Kadaknath chicks. NC= Negative control (No Injection), PC= Positive control (Normal saline treated), ZNP5= ZnONPs5ppm, ZNP 10= ZnONPs 10ppm, VIT. C= Vitamin C.
Significant reduction (P<0.05) of hatchability (56%) was observed in ZnONP 10 ppm treatment (T3
) as compared to positive control group (T1
). Jose et al., (2018)
reported reduction in hatchability due to high levels of zinc, results in imbalance of amnion minerals content that interfered with embryogenesis during incubation or due to the toxicity of zinc nano-form due to its high availability.
Maximum hatchability was recorded in group T4
(vitamin C treatment) as compared to other studied groups. Zhang et al., (2019)
also suggested that increase concentrations of L-Ascorbic acid (L-AA) may improve the hatchability as well as the post hatch performance of chickens. Zhu et al., (2019)
suggested that vitamin C, as a cofactor of hydroxylase, promoting gluconeogenesis and enabling embryos to adapt the environment of incubation and pip of the eggshell.
Non significant increased chick weight and chick’s weight to egg weight ratio was observed in T2
group (P=0.004) where as significant increased (P<0.05) T3
group as compared to positive control group (T1
). Significant decrease (P<0.05) effect on chick growth was observed in T4
(Fig 2b, 2c). The positive effect of ZnONPs 5 ppm supplementation on growth performance was in line with the findings of Torrs and Korvar, (2018)
who recorded the important role zinc in the metabolism of energy, nucleic acids, lipid and protein. In ovo
supplementation of ZnONP 5 ppm solution might be considered as an emerging alternative feed supplement for poultry with a claim of having a greater bioavailability and growth promoter ability. Soltani et al., (2019)
reported improvement in growth performance after in ovo
supplementation of ascorbic acid, should be explained by its antioxidant role.
Oxidative stress and antioxidant status
The perturbation of reactive oxygen species (ROS) and antioxidant balance is often due to an increase in ROS production or/and a depression of the antioxidant system at the time of hatching (Bacau et al., 2021).
The malondialdehyde (MDA) concentration in the chickens hatched from the positive control group (T1
) increased (18.29%) significantly (P<0.05) as compared to negative control group (T0
). In ovo
injected ZnONP 5 ppm (T2
) and Vitamin C (T4
) groups had significant decreased malonaldehyde levels, 21.02% and 25.08% respectively whereas ZnONP 10 ppm treatment recorded a significant increased (P<0.05) levels as compared to the positive control group (Fig 3a).
Fig 3: Effect of in ovo administration of Kadaknath eggs (18 DOI) with normal saline, ZnONP (5 and 10ppm) and vitamin C on (a): Lipid peroxidation; (b): Reduced Glutathione; (c): Antioxidant enzymes, in day old Kadaknath hatchling. NC= Negative control (No injection), PC= Positive control (Normal saline treated), ZNP5= ZnONPs5ppm, ZNP 10= ZnONPs10ppm, VIT. C =Vitamin C.
Our findings are consistent with Zhang et al., (2019),
they also observed that Zn administration can substantially enhance resistance against oxidative stress in developing embryos and hatchlings.
In our study, in ovo
administration of vitamin C significantly reduced the lipid peroxidation levels, which fell into line with the research results of Carr and Maggini, (2017)
. El-Senousey et al. (2018)
recorded that the in ovo
injection of L-AA at various levels increase antioxidant activity and reduces malonaldehyde levels in blood.
Reduced glutathione (GSH) participates in various cellular reactions, scavenges free radicals and maintains redox balance of the cell (Balk et al., 2009).
Significantly reduced (P=0.001) GSH was observed in T1 as compare to the negative control group (T0). Significantly increased GSH levels, 51.25% and 75.0% was recorded in the group treated with ZnONP 5 ppm and Vitamin C respectively whereas significantly reduced (P<0.05) GSH level was reported in group treated with ZnONP 10 ppm (Fig 3b). These results obtained are consistent with the findings of Micheletti et al., (2001)
who revealed that zinc supplementation prevented formation of free-oxygen radicals. Jang et al., (2017)
also observed that dietary supplementation of vitamin C significantly increased total antioxidant status and decreased serum lipid perioxidation in broiler birds.
Fig 3c presented the activity of antioxidant enzymes, Superoxide Dismutase (SOD: E.C. 18.104.22.168) and Catalase (CAT: E.C. 22.214.171.124) in the experimental study. SOD, as an important vitagene is the main driving force in cell/ body adaptation to various stress condition. During embryonic development of the chicken SOD plays a crucial role as an integral part of the antioxidant network (Surai, 2016)
. Significant decreased (P<0.05) activity of SOD in Positive control group (T1
) as compared with T0 group. In ovo
administration of ZnONP 5ppm and Vitamin C reported significant increased (P=0.001) SOD activity, 25% and 43.44% respectively whereas significant reduced SOD activity was observed in T3
as compared to all other treatment groups.
Zinc influences oxidative processes and is also necessary for the structure and function of Cu-ZnSOD, protects tissues from the oxidative lesion (Zhang et al., 2019).
In fact, due to increased antioxidant activity as a result of in ovo
administration of Nano-ZnO 5 ppm, free radicals can be efficiently scavenged and thus the chick embryo was less exposed to oxidative damage during hatching. The increased SOD activity in vitamin C treated groups might be due to the antioxidant action exerted by the enzyme in response to the oxidative stress in Kadaknath hatchlings.
CAT is one of the most important antioxidant enzymes present in almost all aerobic organisms, breaks down two hydrogen peroxide molecules into one molecule of oxygen (Rodriguez-Ruiz et al., 2019)
. Significant decreased (P<0.05) catalase activity in T1
group as compared to negative control group (T0
). ZnONP 5ppm (T2
) and Vitamin C (T4
) treatment had a significant increased catalase (P=0.001) activity as compared to positive control group. ZnONp 10 ppm (T3
) treated group showed significant reduced (P<0.05) catalase activity. Naeem et al., (2022)
also speculated significantly increased catalase activity after in ovo
injection of antioxidants in broiler chickens. Min et al., (2018)
suggested that the high antioxidant activity in the newly hatched chickens can have a positive effect on the growth performance of chickens.
Therefore, the author suggested that the activity of ZnONP 5 ppm and vitamin C following in ovo
injection could perhaps because of its ability to scavenge ROS and alleviating oxidative stress responses.