Effect of Reduced Dietary Copper Levels Sourced from Organic and Nanoparticles Forms on Performance and Nutrient Utilization in Giriraja Birds

Copper (Cu) is an essential inorganic trace element required for growth, production, reproduction and health of animals and birds (Collins et al., 2010). It serves as a cofactor for more than 200 enzymes such as cytochrome oxidase, lysyl oxidase, ceruloplasmin and superoxide dismutase in animal body (Wu et al., 2015). However, the Cu content in major feed ingredients included in poultry diet are unable to meet Cu requirement of birds hence, its supplementation is necessary to maintain the productivity and health of the birds. Further, the absorption and utilization of Cu from conventionally used inorganic sources is poor (Scott et al., 2018). Hence, a large amount of Cu is excreted and its accumulation in the environment causes toxicity in plants and grazing animals (Ferket et al., 2002). Therefore, biotechnological interventions are essential to increase the Cu bioavailability and absorption and reduce its environmental pollution.
       
It is reported that the organic source of Cu can improve its utilization and minimize the level in the excreta and environmental pollution (Nollet et al., 2007) due to its better absorption and metabolism properties. Nollet et al., (2007) also reported no differences in performance of broilers fed diet contained reduced levels of organic chelated Cu but significantly (p<0.05) lower fecal Cu excretion rates. Similarly, nano particles (NP) of Cu having large surface-to-volume ratio, higher bioavailability and less antagonism with other minerals are also better alternatives. As per the current literature available, the Cu-NP is better than its bulk form in enhancing performance of animals (Al-Bairuty et al., 2016). The organic and NP of Cu enhance growth performance, nutrient utilization, reduce pathogen load and improves health of breeder hens (Raje et al., 2018). The dietary Cu was reduced to 50 and 25% of control from organic and NP sources, respectively in Swarnadhara breeder hens without affecting egg production, its quality and hatchability (Aminullah et al., 2021). However, such studies in other improved breeds of birds on growth performance and nutrient utilization are lacking. Hence, the present study was planned to find out the most biologically effective form of Cu with higher efficiency and to reduce its supplementation and excretion level without compromising growth performance and nutrients utilization in Giriraja chicken.

All the procedures with regards to the management and care of the birds followed during the trial were approved by the Institutional Animal Ethics Committee of the University having approval number VCH/IAEC/2020/01, dated 03.03.2020.
       
A genotype akin to country fowl named Giriraja, a multi-colored dual purpose cross breed chicken developed and released by Veterinary College, KVAFSU, Bengaluru for rural scavenging conditions were used. A total of 420-a day-old Giriraja chicks were wing banded and randomly assigned to seven treatment groups of four replicates with 15 chicks in each replicate maintained for 10 weeks of study period under deep litter system with all standard managemental practices. All treatment groups received respective iso-nitrogenous and iso-caloric diets according to their growth stage viz., chick’ diet from 0 to 6 weeks and grower diet from 7 to 10 weeks of age. The daily required amount of feed was weighed and offered replicate wise and recorded. Water was provided ad libitum during the trial period. The experiment was conducted at Veterinary College, Hebbal, Bengaluru, Karnataka, India, located at 13.03^ºN and 77.60^ºE in 2020 for 10 weeks of time period.
       
The Cu nano particles were procured from M/s Matrix Nano Pvt Ltd., New Delhi and inorganic copper sulphate and organic copper as copper proteinate from M/s Zeus-Biotech Pvt. Ltd., Mysuru, Karnataka. The particle size of Cu-NPs was 50-80 nm with 98% purity. The basal diets were formulated as per ICAR (2013) nutrient specifications except Cu. The basal diet was supplemented with 20 ppm Cu in chick phase and 30 ppm in grower phase from inorganic CuSO₄ as control. The treatment diets for chick and grower phases were supplemented with different levels of Cu from organic and NP sources as described in Table 1. The ingredient and chemical composition of basal diets is presented in Table 2. The concentration of Cu in experimental diets (Table 1) was estimated using ICP-OES (Perkin Elmer Optima 8000).
 

 

               
After an adaptation period of 3 days during terminal week of the trial, a total excreta collection method involving two birds in each replicate housed in cage for 3 days was employed to study the bioavailability of different sources of Cu and other nutrients utilization. The samples of feed offered, residue left and excreta voided were analyzed for proximate composition. The nitrogen retained in the body was calculated as the difference between nitrogen intake and nitrogen excreted and expressed as gram per day per kg metabolic body weight (W^0.75) or per cent of intake nitrogen.

The cumulative body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) of birds under different treatments is presented in Table 3. In the study, the BW values of the chick’s stage (0-6^th week) indicated no effect of source and inclusion levels of Cu on chicks BWG. In contrast, during grower phase (7-10^th week) and cumulatively i.e. 1-10^th week, the BWG was significantly (p<0.05) higher in T₂ as compared T₆ and T₇ and control groups. The improved BWG in T₂ treatment group was due to higher (100%) dose and better bioavailability of copper proteinate as compared to inorganic CuSO₄  (Das et al., 2010). Cu plays important role in poultry growth performance which is attributed to its antimicrobial properties that can improve the intestinal health (Usman et al., 2013). In previous reports, the effect of Cu on growth rate was attributed to its function on improved growth hormone axis (Yang et al., 2011) and hypothalamic appetite regulation expression in weanling pigs (Zhu et al., 2011).
 

 
It is hypothesized that Cu-NP due to its novel properties such as large surface area, higher reactivity and better utilization can improve the growth performance and reduce dietary supplementation and excretion rate as well. In the present study, however there is no improved BWG due to Cu-NP, but reduction even up to 75% level had no adverse effect on growth performance as the body weight was comparable to control. The findings are in accordance with those of Sawosz et al., (2018) and Kozlowski et al., (2018) who supplemented Cu-NP reduced by 75 and 80% in broilers and turkey, respectively without observing any reduction in growth performance. The reason is that Cu-NP can more eûectively penetrate cell membrane barriers and can be rapidly distributed independent of the blood circulation (Anjum et al., 2016).
       
No effect of dietary treatments was observed on feed intake during grower phase (7-10 weeks) as well as cumulative period i.e. 1-10 week. In the chick’s stage, the feed intake was significantly lower in T₂ and T₅ as compared to all other treatment groups except of T₇. Despite of reduced feed intake in T₂ and T₅ treatment groups, the BWG was not affected resulting in significant (p<0.05) improvement in feed conversion ratio. During grower phase there was no sighnificant difference in feed consumption among the treatment groups.
       
Cu plays important role in cytochrome C oxidase that contributes for efficient production of adenosine triphosphate and energy metabolism (Wu et al., 2015). Therefore, the better feed conversion ratio at higher Cu level receiving groups (T₂ and T₅) reflects improved energy and nutrients utilization of the diet.
       
The improved gut health due to antimicrobial properties of Cu (Usman et al., 2013) also might have contributed for the better feed conversion ratio. Ramesh (2014) reported that dietary inclusion of Cu-NP at 25 to 50% of requirement can reduce feed intake without compromising the feed conversion ratio in laying hens. However, Wen et al., (2019) reported no significant effect of 20 and 40 ppm CuSO₄ or Cu-methionine on feed intake and feed conversion ratio in broilers.
       
The metabolizability of various nutrients presented in Table 4 indicates significant (p<0.05) increase of DM and CP metabolizability due to Cu supplementation at 30 ppm (organic) and 22.5 ppm (NP). The metabolizability of OM and EE was higher (p<0.05) at 22.5 ppm Cu-NP inclusion as compared to all other groups except for T₂ while CF and NFE metabolizability remained unaffected. The results revealed that the reduced dietary Cu levels sourced from organic or NP has no adverse effect on nutrient utilization. Meanwhile, the highest Cu levels i.e. 100% from organic and 75% from NP sources as compared to standard CuSO₄ were also optimum for nutrient utilization of the diet.
 

       
The reports suggesting effective utilization of nutrients due to Cu antimicrobial properties (Usman et al., 2013), better digestibility of DM, OM and NFE (Gonzales et al., 2009) and greater bioavailability of Cu organic (Nollet et al., 2008) and NP forms (Tamilvanan et al., 2014) are supportive of the finding of the present study. The improved fat utilization can be attributed to the enhanced lipase and phospholipase activities in small intestine with Cu supplementation (Das et al., 2010). The improved nutrient utilization at 22.5 ppm Cu-NP inclusion level confirms its better efficiency as compared to 30 ppm inorganic CuSO₄. However, the results are in contrast to the findings of Sarvestani et al., (2016) who reported no significant effect of 100 ppm Cu-NP dietary inclusion on nutrients digestibility in broilers.
       
Nitrogen balance of experimental birds is presented in Table 5. The source and level of Cu had no effect on total nitrogen intake (g/kg W^0.75/day), while significantly (p<0.05) lower excretion and higher (p<0.05) retention (g/kg W^0.75/day or per cent of N intake) of nitrogen observed at 100% inclusion level from organic and 75% from NP form of Cu as compared to other dietary treatments, however the values were comparable to control.
 

 
In the present study, the inclusion of organic (100%) and NP (75%) form of Cu in relation to CuSO₄ during grower phase was optimum for nitrogen and other nutrients metabolizability which reflect its greater utilization efficiency. The improved nitrogen retention could be speculated that Cu-NP supplementation boost hormone and growth factors in chicken (Das et al., 2010), thereby enhancing protein synthesis and deposition in tissue. The better nitrogen retention can also be associated to the improved energy (OM and EE) metabolizability that can be attributed to better gut health of the chicken. Scott et al., (2016) also reported significantly improved energy and nitrogen utilization and reduction in nitrogen excretion at 20 ppm Cu-NP than CuSO₄ in drinking water. In the present study, however, with reduced Cu supplemental level from organic or NP source, the nitrogen retention and excretion was not adversely affected by the treatment diets as compared to control.
       
Cu balance in the body has been presented in Table 6; indicates that its excretion in dropping in mg per kg W^0.75 per day as well on per cent is significantly (p<0.05) higher in control and linearly reduced in all the treatment groups. Parallel to the excretion, the Cu retention expressed in mg per kg W^0.75 as well on per cent was also found significantly (p<0.05) lower in control and linearly increased in all the treatment groups except for T₃. The excretion was further reduced with reduction of the supplementation either from organic or nano particles of Cu that may be due to minimum Cu supplemental level in the diet.


       
The results are closed to our hypothesis that reduced Cu dietary supplementation level sourced from organic and NP can reduce its excretion rate in dropping to the environment without compromising the chicken performance.
       
These findings do confirm the better efficiency and bioavailability of organic (Zafar and Fatima, 2018) and nano particles (Patra and Lalhriatpuii, 2019) forms of Cu as compared to inorganic CuSO₄ in the diet resulted by reduction in Cu excretion level of droppings to the environment. Similar results were also reported by many studies (Nollet et al., 2007; Gonzales et al., 2009 and Sawosz et al., 2018).

It was concluded that the organic and nanoparticle forms of Cu has dose-independent effect on growth, feed intake, feed conversion ratio, nutrient metabolizability, nitrogen and Cu balance. The dietary inclusion level of Cu can be reduced to 50 and 75% and reduce its excretion level when sourced from organic and nano forms, respectively compared to conventional inorganic CuSO4 without compromising the bird’s performance.

The facilities extended by the Department of Poultry Science, Veterinary College, KVAFSU, Bangalore is duly acknowledged. The first author thanks India-Afghan Fellowship Program, Indian Council of Agricultural Research (ICAR), New Delhi for sponsoring his Ph.D. program.