Indian Journal of Animal Research

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Full Research Article

Effects of Dietary Supplementation of Different Levels of Zinc, Manganese and Copper Nanoparticles on Growth Performance and Bioavailability in Commercial Broilers Chicks

B. Gowtham1, J. Ramesh1,*, R. Karunakaran1, N. Karthikeyan2, P. Raja3
1Department of Animal Nutrition, Madras Veterinary College, Chennai-600 007, Tamil Nadu, India.
2Department of Poultry Science, Madras Veterinary College, Chennai-600 007, Tamil Nadu, India.
3Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India.

ABSTRACT

Background: Nanotechnology is an emerging field and it has shown the great impact in broiler sector for production. Although extensive research is needed to evaluate the safety of nanominerals.  

Methods: A biological study was carried to evaluate the effect of dietary supplementation of inorganic, organic and nano forms of zinc, manganese and copper trace minerals on growth performance and immune status of commercial broiler chicken out for 35 days with 150 numbers of commercial broiler chicken and randomly distributed in 5 treatment groups viz., T1 (Inorganic 100%), T2 (Organic 100%), T3 (Nano 100%), T4 (Nano 50%) and T5 (Nano 25%) comprising of 30 birds in each group.  

Result: The results revealed that body weight, cumulative body weight gain and feed intake of broiler chicken increased significantly (p<0.01) in the 50 and 100% nano trace mineral supplemented groups when compared to other groups without any significant change in FCR. T3 and T4 exhibited significant (p<0.05) improvement in serum superoxide dismutase activity. The mRNA expressions of biomarker gene metallothionein, manganese-superoxide dismutase and ceruloplasmin were upregulated in T3 and T4 groups revealing that the nano trace mineral supplementation had better bioavailability.

INTRODUCTION

Minerals are one of the most important nutrients in the body. The essential minerals are classified into major elements and trace elements depending upon their concentration in the animal body or amounts required in the diet. Some of the essential trace minerals required by broiler chicken are manganese (Mn), iron (Fe), copper (Cu), iodine (I), zinc (Zn), fluorine (F), cobalt (Co) and selenium (Se). These trace minerals account for only 0.3% of the total body mineral concentration (McDowell, 2003). Trace minerals are typically utilised in trace amounts in animals, although difficulties such as decreased bioavailability, antagonistic interactions and higher bodily excretion rates restrict their effectiveness.

Traditionally, trace minerals have been supplemented in animal diets using inorganic salts such as zinc oxide, manganese oxide or sulphate salts and copper sulphate. However, in recent years there are increased concerns about the usage of inorganic trace mineral salts due to their poor bioavailability and potential to cause mineral antagonism, which impairs absorption of minerals and increases environment pollution. Though the organic forms of minerals can be used in smaller amounts than inorganic forms, the higher cost of using organic minerals still discourages from using them.

Nanotechnology is a rapidly evolving science that makes use of materials with dimensions ranging from 1 to 100 nanometres, resulting in novel properties and applications. Nanoparticles, due to their high physical reactivity, can be used as an alternative promoter of health and growth of broilers in much lesser amounts than inorganic forms of these elements, thereby significantly decreasing the excretion of these minerals into the environment. Zinc is a vital trace mineral that is essential for normal metabolism (King, 2011). Manganese is a critical component of several enzymes involved in the defence system against free radicals, protein metabolism and bone formation in the form of superoxide dismutase, ligase, transferase and hydrolases (Keen et al., 2000). Copper is involved in cellular respiration, energy production, collagen synthesis, trace mineral uptake and utilisation, antioxidant activity, heart function, bone formation, tissue keratinization and pigmentation and spinal cord myelination (McDowell, 1992).

MATERIALS AND METHODS

Biological trial and experimental design
 
A total of 150-day-old broiler chicks (Cobb 430) were randomly allocated in a completely randomized design with 5 treatments, each of which has 3 replicates of 10 birds each. Biological trial was conducted for a period of 35 days. The dietary treatments consists of a maize-soyabean meal based diet supplemented with inorganic forms of zinc, manganese and copper at 100% of the requirement as per Bureau of Indian Standards (Bis, 2007) was taken as control (T1), a maize- soyabean meal based diet supplemented with organic forms of zinc, manganese and copper at 100 % of the requirement level (T2), a maize- soyabean meal based diet supplemented with nano forms of zinc, manganese and copper at 100% of the requirement level (T3), a maize- soyabean meal based diet supplemented with nano forms of zinc, manganese and copper at 50% of the requirement level (T4), a maize- soyabean meal based diet supplemented with nano forms of zinc, manganese and copper at 25% of the requirement level (T5).
 
Growth performances
 
Body weights of individual birds were measured early morning before offering feed on days 0, 7, 14, 21, 28 and 35. Cumulative weekly body weight was calculated from the body weight of birds were recorded on day zero and once in every week up to five weeks. Cumulative weekly body weight gain was calculated by subtracting the initial body weight of birds recorded on day one from the cumulative weekly body weight. Cumulative weekly feed intake was calculated by subtracting the quantity of residual feed from feed offered initially. Feed conversion ratio (FCR) was calculated by dividing the total feed consumed in a particular period by body weight gain during that period. Mortality of birds was recorded during the experimental period of five weeks and the per cent livability was calculated.
 
Serum antibody titers against NDV and IBDV
 
Blood samples were collected from 30 birds (six per treatment) on day 21 and 35 of feeding trial. The serum samples were separated. The concentrations of antibodies were analysed by ELISA using IDEXX IBDV Ab Test and IDEXX NDV Ab Test kits, according to the manufacturer’s (IDEXX laboratories, USA) instructions as described by Keck et al., (1993).
 
Superoxide dismutase activity in serum
 
Serum superoxide dismutase activity was measured by the method described by McCord and Fridovich (1969) based on the ability of SOD to inhibit the autooxidation of pyrogallol at pH 8.2. The reaction rate was measured at 420 nm.  
          
Evaluation of Zinc, Manganese and Copper bioavailability  by Real time PCR
 
Metallothionein, Manganese containing superoxide dismutase (MnSOD) and Ceruloplasmin genes were used as biomarkers for zinc, manganese and copper bioavailability utilization in broiler chicken (Varun et al., 2017; Zhang et al., 2020). About 25 mg of liver and muscle tissue samples were homogenized and treated with 750 µl Trizol Reagent (Sigma) to extract the RNA as described by Kaiser et al., (2006). The High-Capacity cDNA Reverse Transcription Kit (Takara Kit) was used for synthesis of cDNA from RNA as per the manufacturer’s instructions The cDNA synthesis cycle conditions were 37°C for 15 minutes, followed by 85°C for 5 seconds. The synthesized cDNA was stored at -20°C until further use. Real-time PCR analysis was performed for the expression of genes (Metallothionein, MnSOD and Ceruloplasmin) and β-actin gene was used as the housekeeping gene. Primers used for Real Time PCR are presented in Table 1. The data were analyzed by the 2-ΔΔCt method and normalized using the expression level of the housekeeping gene. Gene expression was measured by 7500 Fast Real Time PCR (Applied Biosystem Inc, CA, USA) with SYBR PREMIX ex TAQTM (Perfect Real Time, Takara, Shiga, Japan). The thermal cyclic condition used in Real Time PCR was as follows: 95°C for 3 minutes, 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 45 seconds.

Table 1: The sense and antisense primer sequences of target genes and reference gene.


 
Statistical analysis
 
The data obtained from various parameters were grouped and subjected to statistical analysis through the procedure one way Analysis of Variance (ANOVA) using statistical analysis software (IBM SPSS version 22.0 for windows) as per Snedecor and Cochran (1989).

RESULTS AND DISCUSSION

Growth performance
 
The results of the study indicated that body weight (Fig 1), cumulative body weight gain (Fig 2) and feed intake (Fig 3) of broiler chicken increased significantly (p<0.01) in the 50% nano group (T4) when compared to other groups. Body weight gain in birds fed diets supplemented with zinc, manganese and copper in nano forms at 50% requirement level was increased around 10.21% when compared to control group (100% inorganic group).

Fig 1: Effect of dietary supplementation of different forms of zinc, manganese and copper on weekly body weight (g) from 0 to 5 weeks of age in commercial broiler chicken (Mean#±S.E.).



Fig 2: Effect of dietary supplementation of different forms of zinc, manganese and copper on cumulative weekly body weight gain (g) from 0 to 5 weeks of age in commercial broiler chicken (Mean#±S.E.).



Fig 3: Effect of dietary supplementation of different forms of zinc, manganese and copper on cumulative weekly feed intake (g) from 0 to 5 weeks of age in commercial broiler chicken (Mean#±S.E.).


 
Although 100% organic group (T2) and 100% nano group (T3) showed lesser growth and feed intake than the 50% nano group, these groups performed better when compared to 100% inorganic group (T1) and 25% nano group (T5). However, body weight gain in 100% organic group and 100% nano group were increased by 4.11% and 4.92%, respectively when compared to control group. Although the inclusion rate of Zn, Mn and Cu is reduced in 25% nano group, there was no significant difference between 25% nano group and 100% inorganic group in terms of growth performance. However, FCR and per cent livability did not vary significantly between the groups throughout the experimental period (Fig 4). Improvement in growth performance in T4 (50% Nano) over other groups could be attributed to higher bioavailability of Zn, Mn and Cu at reduced inclusion levels and absence of negative interaction among minerals in GI tract. Further, on par growth performance recorded in T5 (25% Nano) compared to control group (T1-100% inorganic) might be due to the better bioavailability of nano minerals compared to their respective mega particles.

Fig 4: Effect of dietary supplementation of different forms of zinc, manganese and copper on cumulative feed conversion ratio and livability (%) from 0 to 5 weeks of age in commercial broiler chicken.


 
Similar results were observed by the other researchers and they reported that supplementation of nano forms of Zn, Mn and Cu resulted in higher body weight gain and feed intake in commercial broiler chicken when compared to their respective inorganic forms and organic forms (El-Katcha et al., 2017; Alkhtib et al., 2020; Matuszewski et al., 2020). They also reported that there was no significant change in FCR.
 
The higher body weight gain observed in nano Zn, Mn and Cu supplemented groups might be due to increased absorption of nano Zn, Mn and Cu from GI tract. Because of smaller particle sizes of Zn, Mn and Cu nanoparticles, a faster diffusion might have occurred in GI tract to reach the intestinal lining. Nano particles with reduced size have greater surface area, thereby they exhibit a higher rate of absorption in the gastrointestinal tract (Hussain et al., 2001). Nano-sized minerals distribute quickly from the circulation to the tissues and they are mostly targeted at organs with a well-perfused reticuloendothelial system (RES), such as the spleen and liver (Geraets et al., 2014).
 
Serum antibody titers against NDV and IBDV
 
The results of the present study indicated that there was no significant difference between the treatment groups in antibody titer value against NDV after 14th day of vaccination. After 28th day of vaccination, the control groups had significantly higher titer values against NDV when compared to all the other groups (Fig 5).

Fig 5: Effect of dietary supplementation of different forms of zinc, manganese and copper on serum antibody titer value against NDV and IBDV in commercial broiler chicken (Mean#±S.E.).



The results of the present study were in accordance with Sabaghi et al., (2021) who found that the nano manganese supplementation at 100 ppm and 150 ppm levels in broiler chicken showed reduced (p<0.05) antibody titer values against NDV when compared to the inorganic manganese supplementation.
 
Superoxide dismutase activity in serum
 
Superoxide dismutase activity in broiler chicken is influenced by the supplementation of different forms of Zn, Mn and Cu. In the present study, a significant increase in serum SOD activity was observed in 100% nano (T3) and 50% nano (T4) groups when compared to other treatment groups. However, no difference was observed between 100% inorganic group (T1) and 25% nano group (T5) (Fig 6).

Fig 6: Effect of dietary supplementation of different forms of zinc, manganese and copper on serum superoxide dismutase activity on 35th day of age in commercial broiler chicken.



Similar results of increased SOD enzyme activity in plasma were reported by Abedini et al., (2018) with the ZnO nanoparticle supplementation in broilers when compared to the control group. El-Kazaz and Hafez, (2020) concluded that broilers fed with copper nanoparticles have considerable increase (p<0.05) in serum copper and SOD levels compared to CuSO4 treated birds. It was reported that Zn, Mn and Cu nanoparticles were found to have higher bio-availability than their inorganic and organic counterparts because of their faster rate of absorption (Jankowski et al., 2020).
 
Bioavailability of biomarkers of zinc, manganese and copper
 
The results of the present study indicated that the metallothionein gene expression in liver and muscle was higher in 100 and 50 % nano mineral supplemented groups when compared to other treatment groups (Fig 7). Similarly, Varun et al., (2017) reported that the broiler chicken fed with nano zinc had relatively higher expression of metallothionein gene in liver when compared to inorganic zinc.

Fig 7: Effect of dietary supplementation of nano forms of zinc, manganese and copper on their relative gene expression in liver and muscle using real time PCR.



MnSOD mRNA levels in both liver and muscle were higher (p<0.01) in 100% and 50% nano mineral supplemented groups when compared to inorganic mineral supplemented group. Similarly, Zhang et al., (2020) reported that the dietary supplementation of manganese improved the gene expression of MnSOD thereby increasing the intestinal immunity in broiler chicken.

Liver ceruloplasmin mRNA levels were not influenced by dietary copper supplementation of inorganic, organic or nano copper in broilers. Accordingly, Scott et al., (2018) reported that ceruloplasmin mRNA gene expression was not affected by dietary source of copper in liver tissue of broiler chicken. Hence, liver ceruloplasmin mRNA expression might not be an efficient biomarker to indicate copper bioavailability. However, significant upregulation was observed in muscle ceruloplasmin mRNA expression when the birds are supplemented with organic and nano forms of copper at 100 and 50% requirement levels.

It was revealed from this study that the supplementation of nano forms of zinc and manganese upregulated the expression of metallothionein and MnSOD in liver and muscle tissue. Whereas, nano supplementation of copper upregulated the expression of ceruloplasmin in muscle tissue. These upregulation of biomarkers for zinc, manganese and copper indicated that the nano form of supplementation had increased the bio-availability of these three trace minerals in broiler chicken.

CONCLUSION

The supplementation of nano zinc, manganese and copper at 25% requirement level was sufficient to raise the broiler chicken without any deficiency symptoms. The growth performance was at par with the birds fed with diets supplemented with inorganic forms of zinc, manganese and copper at 100% requirement level. However, improved growth performance was observed in nano zinc, manganese and copper supplemented group at 50% requirement level.

ACKNOWLEDGMENT

The authors thank Tamil Nadu Veterinary and Animal Sciences University for providing the necessary facilities to carry out the research work.

CONFLICT OF INTEREST

None.

REFERENCES


  1. Abedini, M., Shariatmadari, F., Torshizi, M.A.K. and Ahmadi, H. (2018). Effects of zinc oxide nanoparticles on performance, egg quality, tissue zinc content, bone parameters and antioxidative status in laying hens. Biological Trace Element Research. 184(1): 259-267.

  2. Alkhtib, A., Scholey, D., Carter, N., Cave, G.W., Hanafy, B.I., Kempster, S.R. and Burton, E.J. (2020). Bioavailability of methionine -coated zinc nanoparticles as a dietary supplement leads to improved performance and bone strength in broiler chicken production. Animals. 10(9): 1482.

  3. BIS, (Bureau of Indian Standards) (2007). Indian Standard Poultry Feeds- Specification. 5th Edn. New Delhi.

  4. El-Katcha, M., Soltan, M.A. and El-Badry, M. (2017). Effect of dietary replacement of inorganic zinc by organic or nanoparticles sources on growth performance, immune response and intestinal histopathology of broiler chicken. Alexandria Journal for Veterinary Sciences. 55(2).

  5. El-Kazaz, S.E. and Hafez, M.H. (2020). Evaluation of copper nanoparticles and copper sulfate effect on immune status, behavior and productive performance of broilers. Journal of Advanced Veterinary and Animal Research. 7(1): 16.

  6. Geraets, L., Oomen, A.G., Krystek, P., Jacobsen, N.R., Wallin, H., Laurentie, M. and de Jong, W.H. (2014). Tissue distribution and elimination after oral and intravenous administration of different titanium dioxide nanoparticles in rats. Particle and Fibre Toxicology. 11(1): 1-21.

  7. Hussain, N., Jaitley, V. and Florence, A.T. (2001). Recent advances in the understanding of uptake of. Advanced Drug Delivery Reviews. 50: 107-142.

  8. Jankowski, J., Otowski, K., Kozłowski, K., Pietrzak, P., Ferenc, K., Ognik, K. and Zduñczyk, Z. (2020). Effect of different levels of copper nanoparticles and copper sulfate on morphometric indices, antioxidant status and mineral digestibility in the small intestine of turkeys. Annals of Animal Science. 20(3): 975-990.

  9. Kaiser, M.G., Cheeseman, J.H., Kaiser, P. and Lamont, S.J. (2006). Cytokine expression in chickenperipheral blood mononuclear cells after in vitro exposure to Salmonella enteric serovar Enteritidis. Poultry Science. 85(11): 1907-1911.

  10. Keck, L.D., Skeeles, J.K. and McNew, R.W. (1993). Antibody detection in matched chicken sera and egg-yolk samples by commercial enzyme-linked immunosorbent assay kits for Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus and avian reovirus. Avian Diseases. 825-828.

  11. Keen, C.L., Ensunsa, J.L. and Clegg, M.S. (2000). Manganese metabolism in animals and humans including the toxicity of manganese. Bio Metals. 70(12): 1391-1393

  12. King, J.C. (2011). Zinc: an essential but elusive nutrient. Am. J. Clin. Nutr. 94(2): 679S-684S.

  13. Matuszewski, A., £ukasiewicz, M., £ozicki, A., Niemiec, J., Zieliñska- Górska, M., Scott, A., Chwalibog, A. and Sawosz, E. (2020). The effect of manganese oxide nanoparticles on chicken growth and manganese content in excreta. Anim. Feed Sci. Technol. 268: 114-597.

  14. McCord, J.M. and Fridovich, I. (1969). Superoxide dismutase: An enzymic function for erythrocuprein (hemocuprein). Journal of Biological Chemistry. 244(22): 6049-6055.

  15. McDowell, L.R. (1992). Minerals in Animal and Human Nutrition, Academic Press Inc. San Diego, New York. pp. 524.

  16. McDowell, L.R. (2003). Minerals in Animal and Human Nutrition (2nd Edn). Elsevier Science BV.

  17. Sabaghi, S., Razmyar, J. andHeidarpour, M. (2021, October). Effects of nano-manganese on humoral immune response and oxidative stress in broilers. Veterinary Research Forum. 12(4): 487-491.

  18. Scott, A., Vadalasetty, K.P., £ukasiewicz, M., Jaworski, S., Wierzbicki, M., Chwalibog, A. and Sawosz, E. (2018). Effect of different levels of copper nanoparticles and copper sulphate on performance, metabolism and blood biochemical profiles in broiler chicken. J. Anim. Physiol. Anim. Nutr. 102(1): 364-373.

  19. Snedecor, G.W. and Cochran, W.G. (1989). Statistical Methods, 8th Edn. Ames: Iowa State Univ. Press Iowa. 54: 71-82.

  20. Varun, A., Karthikeyan, N., Muthusamy, P., Raja, A., Ruban, S.W. and Tamilkumaran, J. (2017). Carcass Characteristics and bone measurements of commercial broilers fed with inorganic, organic and nano Zn containing diets. Int. J. Curr. Microbiol. App. Sci. 6(9): 3748-3756.

  21. Zhang, H., Pan, S., Zhang, K., Michiels, J., Zeng, Q., Ding, X. and Bai, S. (2020). Impact of dietary manganese on intestinal barrier and inflammatory response in broilers challenged with Salmonella typhimurium. Microorganisms. 8(5): 757.
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