Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Effects of in ovo Administration of Zinc Oxide Nanoparticles and Vitamin C on Hatchability Performance and Redox Status in Day Old Kadaknath Hatchlings

M.K. Kesariya1, M. Roy1, O.P. Dinani2, S. Dora3, S. Roy4, S. Prusty5
1Veterinary Physiology and Biochemistry Department, College of Veterinary Science and Animal Husbandary, Durg-491 001, Chhattisgarh, India.
2Instructional Livestock Farm complex College of Veterinary Science and Animal Husbandary, Durg-491 001, Chhattisgarh, India.
3Subject Matter Specialist Krishi Vigyan Kendra, Jagdalpur-491 001, Chhattisgarh, India.
4Veterinary Medicine Department, College of Veterinary Science and Animal Husbandary, Durg-491 001, Chhattisgarh, India.
5Animal Nutrition Department, College of Veterinary Science and Animal Husbandary, Durg-491 001, Chhattisgarh, India.
Background: In poultry industry, hatcheries play a vital role in connecting the poultry production chain and are expected in the productive performance, with an impact on company profits. The use of in ovo feeding support poultry embryonic development and offers the production efficiency and welfare of commercial poultry. 

Methods: This study investigated the impact of in ovo administration of normal saline, Zinc oxide nanoparticles and Vitamin C on hatchability, chick growth and redox status in Kadaknath hatchlings. Zinc oxide nanostructures were synthesized by chemical method and characterized for size determination. A total of 150 fertile eggs of the Kadaknath poultry breed were divided into five groups (T0 to T4) and treated with in ovo administration of 200 μl each of normal saline, zinc oxide nanoparticles (5 and 10 ppm) and Vitamin C respectively on the 18th day of incubation through air sac into amniotic fluid.

Result: Rod shaped nanostructures ranging from 45 to 98 nm were synthesized and showed sharp peak positioned at 436.59 cm-1. Zinc nano composite 5 ppm and vitamin C administration had significantly (p<0.05) improved hatchability, hatch weight, chick weight and egg weight ratio and antioxidant status. 
The hatchery performance in poultry sector play a vital role in connecting the poultry production chain with an impact on company profits (Desha et al., 2015). Kadaknath is the most popular native chicken breed in the Jhabua district of Madhya Pradesh, India. Geographical Indication Registry and Intellectual Property India awarded Geographical Indication (GI) Tag to this chicken breed on 30 July 2018 (GI Journal, 2018). This breed is experiencing higher demand due to its unique characteristics and perceived health benefits of its meat and eggs (Haunshi and Prince, 2021). In ovo administration of nanominerals, acting as bioactive agents and carriers of nutrients may be seen as a new method of nano nutrition, providing poultry embryos with bioactive compounds and/or with an additional quantity of nutrients and energy (Palouj et al., 2021). Antioxidant capability at hatching time is considered to be an important determinant of chick viability and prevent the damaging effects of free radicals and toxic products of their metabolism (Dominguez et al., 2019).Thus, the use of in ovo feeding of antioxidant constituents may be of immense benefit to the embryo in the pre-hatch growth phases (Asa et al., 2022). Thus, the objective of this study was to investigate the effect of in ovo administration of Nano zinc and vitamin C on hatchability performances and antioxidant status in day old Kadaknath hatchlings.
The present study was conducted from April, 2021 May, 2022 at the Department of Veterinary Physiology and Biochemistry and Poultry unit, College of Veterinary Science and A.H., DSVCKV, Anjora, Durg (C.G.). The research was intended to use Zinc Oxide nanoparticles (ZnONPs) and Vitamin C on hatchability, redox status, in Kadaknath hatchlings.
Synthesis and characterization of zinc oxide nanoparticles (ZnONPs)
The ZnONPs were synthesized by using zinc nitrate hexahydrate (Zn(NO3)2.6H2O) and sodium hydroxide (NaOH) precursors, calcined at 400°C  as per method described by Fiedor-Tobola et al. (2018) and characterized by Fourier-transform infrared spectroscopy (FTIR) (Bruker, Vertex 80 FTIR System Germany, IIT Madras) and Scanning Electron Microscopy (SEM) (ZeissEVO18, IIT Delhi).
Experimental design
A total of 250 embryonated eggs of 8-9 month-old Kadaknath hens, laid on the same day represented the same weight class was procured from Poultry Unit, College of Veterinary Science and A.H., Anjora, Durg (Chhattisgarh)-491001 India. The basal diets of for breeder birds were formulated as per ICAR. (2013) with nutrient specifications for poultry (Metabolizable energy 2700 Kcal/Kg). The eggs were disinfected, weighed, numbered and incubated with the temperature and relative humidity at 37.8°C and 55% respectively. On the18th day of incubation (18 DOI) infertile eggs and eggs containing dead in-shell embryos were removed. A total of 150 embryonated eggs were individually weighed and randomly assigned to 5 groups with 3 replicates in each and 10 eggs per replicate. The T1 was kept as healthy (Negative) control (without injection), the T2, T3, T4 and T5 received in ovo treatment (200 μl) of normal saline (positive control), zinc oxide nanoparticle 5 ppm (ZnONP5 ppm solution in normal saline), zinc oxide nano particle 10 ppm (ZnONP10 ppm solution in normal saline) and Vitamin C (Mankind Pharma Ltd, 150 mg/1.5 ml) respectively through the air sac into amniotic fluid using a 21-gauge needle and eggs were sealed with sterile tape and placed in an incubator. On 21 DOI and 22 DOI, the number of eggs hatched was recorded and all hatchlings were weighed.
Hatchability and growth performance of chicks
Per cent hatchability, hatch weight of chicks and chick weight to egg weight ratio per cent was determined:
Sample collection and biochemical analysis
Blood samples were collected from randomly selected day-old hatchlings per group (3 per replicate) and hemolysate was prepared as per method described by Huntsman, (1975) to estimate lipid peroxidation and antioxidant status. The haemoglobin (Hb) content of the erythrocytes hemolysate was determined by the cyanmethemoglobin method.
Evaluation of oxidative stress and antioxidant balance
The LPO concentration in the erythrocytes was determined as per method describe by Utley et al., (1967) and expressed in nmol MDA/mg of Hb. Reduced glutathione (GSH) assay was performed according to Prins and Loos (1969) and determined as µmol GSH/mg of Hb. The activity of superoxide dismutase (SOD) was measured by using nitroblue tetrazolium and was expressed as in Unit/mg of Hb. (Marklund and Marklund, 1974).The activity of catalase (CAT) in Unit/mg of Hb was assessed as described by Cohen et al., (1970).
Statistical analysis
The collected data were subjected to statistical analysis using the general linear model of one-way analysis of variance - ANOVA. The significance of differences between the groups was evaluated using Tukey’s test (IBM SPSS Statistics version 26.0).The level of significance was set at p≤0.05. Simplifying the visualization of these results, depicted interleaved bars±SEM.
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 performance
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).
Lipid Peroxidation
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. 
Antioxidant status
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. and Catalase (CAT: E.C. 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. 
The results of the present study showed that in ovo administration of Nano-ZnO 5 ppm(45 to 98 nm size) and vitamin C on the 18th day of incubation substantially enhanced antioxidant capacity in hatchlings of old Kadaknath hatchlings.
The authors declare that they have no competing interests.

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