Effects of Dietary Supplementation of Zinc Oxide Nanoparticles on Productive Performance, Physiological, Histological Changes and Tissues Zn Concentration in Broiler Chicks

doi:10.21203/rs.3.rs-1523659/v1

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Effects of Dietary Supplementation of Zinc Oxide Nanoparticles on Productive Performance, Physiological, Histological Changes and Tissues Zn Concentration in Broiler Chicks

https://doi.org/10.21203/rs.3.rs-1523659/v1

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A five weeks biological experiment was planned to examine the impacts of dietary supplementation with of Nano sized of zinc oxide (ZONPs) on productive performance, carcass traits, organ relative weights, serum biochemical parameters, histological alteration in some internal organs and concentration of this element in the serum, liver, thigh and breast muscle in broiler chicks. A total of 108 day old newly hatched unsexed commercial broiler chicks were individually weighed and equally distributed in a completely randomized design arrangement according to the dose of zinc oxide nanoparticles (ZONPs) supplementation into 3 dietary experimental groups, with 3 replications having 12 birds per replicate (n = 36/ treatment) with feed and water were provided ad libitum consumption through 5 weeks life span. The 3 Experiential dietary treatments (T1, T2 and T3) received corn-soybean meal-based diets enhanced with 0 (control), 40 and 60 mg / kg diet of ZONPs respectively.

Present results has shown that after 5 weeks of feeding trial and as compared to control, the ZONPs supplementation groups recorded higher body weight, improved feed consumption, feed conversion ratio and performance index. Serum biochemical analyses revealed that serum cholesterol, triglyceride, low density lipoprotein and uric acid decreased significantly, while high density lipoprotein and liver enzyme (AST,ALT) concentrations were significantly increased in ZONPs groups than control. Meanwhile, zinc accumulation in serum, liver and breast and thigh muscle were linearly increased with increasing zinc supplementation. It could be concluded that supplementation of ZONPs to broiler diet at 40 or 60 mg/kg improved productive performance, birds' physiological status and the lower levels Zn (40 mg/kg diet) revealed promising results and can be used as an effective feed additive in broilers.

Zinc oxide nanoparticles

performance

physiological

tissue concentration

broiler

Poultry industry is an important economical business and fastest growing sector in the agriculture field, which holds the primacy in meeting the needs of the population in meat and eggs and its processed products that increase the nutritional quality of human food. It has become an attractive business due to its rapid outcomes [1]

Full and balanced diet of poultry with high-quality feeds and availability of minerals is the key to high safety and productivity of animals. The needs of animals for minerals can be met through various forms of compounds: inorganic, organic, chelated and nanoscale.

Zinc (Zn) is a highly important trace mineral with widely varying functions and biological activities in many physiological, metabolic and digestive processes in birds [2,3]. It acts as a cofactor of more than 300 enzymes which essential for reproduction and protein, lipid and carbohydrate metabolism[ 4], appetite control, growth, glandular development [5]. Furthermore, it's necessary for hormone secretion i.e. growth and insulin hormones [6,7], nucleic acid synthesis, gene expression, cellular division, signal transduction, gene transcription [8], protein synthesis, and wound healing [9,10]. Moreover it is responsible for activation of the antioxidant status, its vital free radical’s scavenger of the antioxidant defense system [11-13].

The National Research Council recommended [14] level of Zn 40 mg/kg of diet, on the other hand, with continued genetic modification of commercial broiler strains, this level is no longer provided sufficient growth, health and production for modern broiler strains. Consequently, poultry producers added these salts to the diets at levels higher than recommended by NRC to meet bird requirements[15], neglecting that high Zn supplementation results in increased production costs and an increases mineral excretion in feces resulting in environmental pollution especially at the large scale of breeding [16]. In addition, higher inclusion levels of Zn may affect the balance of other microelements and reduce the stability of vitamins and other nutrients and increase its accumulation inside the animal body [13].

Traditionally Zn is usually supplemented in the poultry feed in the inorganic forms such as sulfates, oxides and chloride due to its lower cost and commercial availability. However, due to the pH changes that naturally occur in the digestive tract of poultry, there may be antagonism and interactions among trace minerals, as well as with other compounds in diet as phytic acid found in most of the grains based diets forming insoluble compounds in the gastrointestinal tract, preventing their absorption in body thus impair zinc absorption [17].

Many authors documented that the inorganic trace minerals may be replaced by organic sources to reduce the over supplementation and decrease their excretion [18,19]. The supplementation of organic Zn in broiler diets is limited until now due to its relatively high cost [13]. Therefore, enhancement of Zn bioavailability may help to solve such problems.

The use of Nano particles of mineral elements, represents a promising and emerging technology in the animal farming industry. Nano particles of mineral have higher bioavailability, because of their novel characteristics, such as, greater specific surface area, a lot of surface active centers, higher surface activity, high catalytic efficiency and stronger adsorbing ability [20,21], as well as it can be easily carried up by the gastrointestinal tract and further distribute into the blood and utilized in the animal system and reached too deeper tissues by more efficient than the larger sized particles [22]. Researchers have used from nanotechnology science and those products as additives in poultry nutrition to achieve positive effects in poultry production, some of products is zinc nanoparticles [23].

Zn are commercially produced in nanoparticles forms called Nano Zn. Nanoparticles of Zinc oxide is considered as an emerging alternative feed supplement for poultry with a claim of having a greater bioavailability and exerting a superior efficacy compared with both inorganic and organic forms [24-26]. Nano-Zn may be used at lower doses and confer more beneficial effects for both livestock and the environment [13,25, 27].

It has been indicated in several studies that Nano-zinc outweighed the conventional zinc sources, at same or even lower doses, in terms of productive and reproductive performance ,carcass traits, bone development, antioxidant defense and act as high antibacterial agent, antifungal, and growth promoter ability [28-32]. Also, improving the GIT microbial population, cell repair and enhancing the immune system [13,33].

Accordingly, the present work was designed to investigate the effect of two levels of zinc oxide nanoparticles supplementation in diets on growth performance, carcass traits, serum biochemical profile, histological alteration, and tissue concentrations of Zn in broiler chicks.

Animal ethical approval

This experimental biological study was conducted for a period of 5 weeks at the experimental animal house and laboratory belonging to poultry physiology and production researches unit, Biological Application Department, Nuclear Research Center, Atomic Energy Authority, Egypt.

All of the experimental procedures, techniques, material and methods used throughout this study regarding birds welfare, housing, treated, handling during experimentation were done and approved in accordance with guidelines of the local ethics committee for care and use of animal in a humane manner by the Institutional Council of the Biological Application Department, Nuclear Research Center, Egypt.

Birds accommodation, husbandry, diets and experimental design

A total number of one hundred and twenty, day old newly hatched unsexed broiler chicks (Cobb) obtained from a local hatchery were housed in batteries and fed recommended diets without any treatment till the third day of age as the adaptation and quarantine period to confirmed that they were free of pathogen and any other disease.

At the third day of chicks age (experimental initiation), all chicks were weighed individually and the upper and lower body weights were withdrawn. The remainder 108 chicks were used in this study from 3 day to the end of experiment at 5 weeks of age (experimental termination). The chicks were weighed individually (initial weight) and randomly distributed equally in number in cages in a completely randomized design into 3 different dietary treatment groups (similar in average body weight) according to the dose of zinc oxide nanoparticles (ZONPs) supplementation. Each treatment consisted of 36 chicks which further subdivided into 3 replicates (cages) having 12 chicks in each. All replicates were separately housed by positioning at random in cages batteries in the same room. The experimental dietary treatments included the un-supplemented basal diet with ZONPs (T1 control), and supplemented with 40mg ZONPs /kg diet (T2) and 60 mg/kg diet of ZONPs (T3).

chicks were housed and reared in wire-mesh floor stainless steel clean galvanized batteries cages in an environmentally controlled open – sided house under the same uniform management, standard hygienic and environmental conditions. The batteries were provided by suitable feeder and fresh water via stainless steel drip nipple drinkers, which were adjusted continuously with the age of the birds, provide sufficient feeding and watering space for the birds and allowing the birds to be unrestricted (ad libitum) access to feed and water during the experimental period. Chicks received a continue lighting program of 24 h light / day for first 3 day and 23 h light:1h dark cycle per day until the experiment termination. House temperature was adjusted using electric heaters and maintained at 34°C during the first 3 d of life and then was reduced gradually 2-3 °C weekly until reaching 24°C at the end of 5^th week and the vaccination program was carried out during the experiment as per schedule.

The experimental diets were formulated and varied according to feeding stage and in between groups in ZONPs levels. Birds were fed according to 3-phase feeding period; for starter period (0-12 day of age; contained % CP and Kcal/ ME kg diet), and subsequently grower ( 13-28 day; contained % CP and Kcal/ ME kg diet) and finisher ( 28-35 day ; contained % CP and kcal / ME kg diet). All experimental diets was fed in mash form and the nutrient content of the diets was calculated based on the chemical composition of the feedstuffs according to the equations of formulated to the nutrient specifications of the Cobb broiler strain which was modified to meet or exceed the feeding standards of the NRC (1994) requirements for broilers except for Zn. The experimental diet composition and chemical analysis was done in the laboratories and presented in table (1).

Table (1) the ingredient composition and calculated chemical analysis of the basal diet.

Ingredient composition(kg)	starter	grower	finisher
Yellow corn	56	59	64
Soybean meal 44%	39.5	35	29
Vitamin and mineral Premix	0.3	0.33	0.33
Monocalcium phospate	1.5	1.4	1.3
Sodium chloride	0.2	0.2	0.2
Bicarbonate	0.2	0.2	0.2
Vegetable oil	0.6	2.2	3.3
limestone	1.1	1.1	1.1
Methoinin	0.3	0.3	0.3
Lysin	0.2	0.16	0.18
Cholin chloride	0.18	0.15	0.15
Calculated chemical analysis
Crude protein %	22.14	2.42	18.2
Metabolizable energy	2811	2955	3088

Vitamin-mineral premix provided per kg of diet: vitamin A, 14,000 IU; vitamin D3, 3,000 IU; vitamin E, 34.5 IU; vitamin K3, 4 mg; biotin, 0.15 mg ;vitamin B1, 1.3 mg; vitamin B2, 6 mg; pantothenic acid, 20 mg; vitamin B6,3 mg; niacin, 60 mg; vitamin B12, 6 mg; choline, 150mg; folic acid,0.5 mg. Mn, 60 mg; Fe, 50 mg; Cu, 10 mg; I, .1.5 mg; Se, 0.2 mg; Zn, 0 mg.

Zinc oxide nanoparticles (ZONPs) preparation, synthesis and purification.

ZONPs were synthesized according to the method described by [34] using the cell free culture filtrate of the fungus Alternaria tenuissima AUMC10624. (Culture Collection of Assiut University Mycological Center, Assiut, Egypt, http://www.aun.edu.eg/aumc/aumc.htm). Fungal spores from the cultures (7 days old) of A. tenuissima were harvested and the spore concentration was adjusted to a concentration of 10⁶ spores/ ml. Spore suspension was transferred in vial sealed with paraffin and irradiated by gamma rays at a dose of 500 Gy (the best irradiation dose for maximum synthesis of ZONPs). Irradiation process was carried out at the Nuclear Research Center (Cairo, Egypt) using ⁶⁰Co Gamma chamber, MC20, Russia, with an average dose rate of 605.726 Gy h^-1 at the time of the experiment.

After irradiation, 1 mL of the irradiated spore suspension was added to 250 mL flasks containing 50 mL medium. The flasks were incubated for 10 days at 30 °C and the ATCF was used for the production of ZONPs.

Synthesis, separation and purification of ZONPs:

A volume of 100 mL of the A. tenuissima cell-free culture filtrate (ATCF) was taken in an Erlenmeyer flask and mixed with 100 mL of 2mM zinc sulfate (Zinc sulfate was purchased from Sigma-Aldrich (St Louis, MO, USA). The reaction mixture was maintained under vigorous stirring for 20 min at room temperature and the mixture was observed visually for formation of white precipitate. The precipitate (ZONPs) was separated by the ultra-centrifugation for 20 min at 20000 rpm. After which, the separated nanoparticles were washed in deionized water and ethanol and then dried at 50°C in a hot air. Finally, the fine powder of ZONPs was dissolved in ethanol (HPLC grade) and treated ultrasonically for the dispersion of the individual nanoparticles and used. Dynamic light scattering and Zeta potential analyses (Zetasizer Nano ZS, Malvern instruments, Worcestershire, UK) were carried out to estimate the distribution of different size particles dispersed in ZONPs solution and its stability. The obtained results were in complete agreement with the study concerning the synthesis of ZONPs[34]

Sampling and measurements

Productive Performance

Production parameters were evaluated for each replicate in terms of live body weight (LBW), feed consumption (FC) feed conversion ratio (FCR), mortality and performance index (PI). Broiler chicks were weighed individually to the nearest gram at the beginning of the experiment and then weekly intervals until the end of the experiment to evaluate weekly LBW. Mortality was observed and recorded daily .Feed consumption was recorded weekly and adjusted for mortalities until the end of the experiment. Adjusted FCR was calculated at the end of experiment for each group as gm feed / gm body weight, considering the weight of dead birds. Also, the performance index (PI%) was calculated on the basis of [35]:-

PI= Live body weight in gm / feed conversion X100

Carcass characteristics, organs index and blood biochemical analysis

At the end of experimental period, 9 birds of each group (3 birds from each replicate) were chosen based on average body weight in each cage for both blood sampling analysis and removed digestive organs to determine carcass characteristics. Chicks were deprived from feed overnight with free access to water to decrease the direct effect of feed on blood parameters before slaughtering. After feed deprivation, birds weighed individually, slaughtering, defeathering and carcass weight was determined after removal of feathers, feet, head, and internal organs. The eviscerated carcass yield, and internal organs (liver, kidneys, gizzard, proventriculus, heart, and intestine) were extracted, blotted to dry, weighed individually and calculated as a percentage of fasted live body weight. At the same time, liver, kidney and intestine were used for histological study.

During bleeding, individual blood samples were collected from birds into a sterile dry clean centrifuged tube without anticoagulant, and then kept at room temperature for one hour to allow clotting of blood. After which the clot was rotated gently away from the tube wall using a clean fine metal wire, Separation of serum was carried out by centrifugation of coagulated blood at 3000 rpm for 10 minutes. The clear serum was carefully harvested and transferred to dry sterile screw capped tubes (Eppindrof tube) and stored at -20 ºC for subsequent chemical analyses.

serum constituent of glucose and cholesterol (chol), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein(LDL) as a lipid profile assay and aspartate amino transferase (AST), alanine amino transferase (ALT) as a liver function and uric acid and creatinine (creat) as a kidney functions were calorimetrically analyzed using commercial analytical kits (Bio-diagnostics) according to the procedures outlined by the manufacturers and were determined by SHIMADZU UV 1601 spectrophotometer.

Estimation of tissue and serum zinc concentrations (ppm)

Tissues and serum zinc concentrations were determined by inductively coupled plasma optical emission spectrometer (Leeman Prodigy High Dispersion ICP-OES,USA) at the nuclear research center, Egyptian atomic energy authority. The registered values for zinc were expressed as (ppm).

At the end of the experiment, samples from liver, breast and thigh muscle and serum were collected from the same 9 birds per each group. Briefly, 0.5g of tissue samples were oven dried at 100°C for 24 h, then exposed to digested with 5 mL of concentrated HNO3 and 2 ml of H2O2 in a 50-mL calibrated flask at 120°C under a heating plate until the solution became clear . The content were further digested using 5 mL of concentrated HNO3 under a heating plate at 100 °C. A blank and the digested samples were then allowed to cool and ﬁltered through Whatman 42 filter paper. The 1 ml extract was then diluted using deionized water to the required volume. To determine serum Zn concentration, the procedure was the same as that of the tissues analysis but with using 1 ml of serum sample.

Histological examination:

Liver, kidney and intestine from the same 9 birds slaughtered were selected for histological studies to evaluate the presence of histopathological lesions. The procedure for histological preparation is as described by [36].Stained sections were examined using Leica microscope (CH9435 Hee56rbrugg) (Leica Microsystem, Swithereland) in Faculty of Veterinary Medicine, Cairo University.

Statistical analysis

Observed data were subjected to statistically analyzed by one way analysis of variance (ANOVA) as a completely randomized design using the general linear models (GLM) procedure under statistical analysis system software [37]. Significant differences among the means were determined by using Duncan’s multiple-range test [38] at P < 0.05. The data are presented as means ± standard error (SEM).

Growth performance

There was no mortality and the general health status of broilers in all groups was good throughout the experimental period.

The overall growth performance results are shown in Table (2 and 3), revealed beneficial effects for supplementation ZONPs to broiler diets. Table (2) summarized the effect of dietary ZONPs supplementation at two experimental levels on body weight of broiler chicks. It could be noticed that the birds had approximately the same initial body weight at the beginning of the experiment in all dietary treatments. During the whole experimental weeks, the chicks fed on dietary ZONPs supplementation at both level had the heaviest live body weight, in comparison with those fed on control diet, with notice that T2 was the highest value followed by (T3) as compared with the T1 (control) at all experimental weeks.

The effects of dietary ZONPs supplementation on feed consumption (FC), cumulative feed conversion ratio (FCR) and performance index (PI) are presented in Table (3). Results showed that the amount of feed consumed (FC) per chicks per week was affected by dietary treatments. Addition of ZONPs in broiler diets (T2 and T3) resulted in numerical increased in weekly and cumulative feed consumption than the control (3.257 3.537 and 3.437 for T1, T2 and T3, respectively). Concerning feed conversion ratio (FCR), the data presented in Table 3 indicated that the quantity of feed required per unit of weight was increased in ZONPs - treated birds as compared with control. The FCR was 1.537 and 1.570 for T2 and T3 respectively compared with 1.625 for T1. At the same time, the same trend was also observed in related to performance index (PI), where performance index was increase in broiler chicks fed a dietary supplementation with ZONPs than the control chicks.

Carcass characteristics and relative organ weights

Carcass traits and internal organs weight relative to body weight of broiler chicks, as affected by dietary treatment are tabulated in table 4. Statistical analysis (P < 0.05) showed that supplemented broiler diets with ZONPs (T2 and T3) caused a significant increase in carcass yield as compared with control group (T1). ZONPs at 40 mg/kg increased carcass yield in comparison with other groups. Results in the same table also indicated that dietary ZONPs supplementation (T2 and T3) significantly decreased relative kidney, proventriculus and intestine weights. On the other hand, liver weight was increased at both supplemented levels than control, but was significantly only in T3 chicks with increasing ZONPs dose. The relative weight of gizzard in T2 birds only was significantly (P<0.05) lower than birds fed on the control one and no significant difference was observed when compared T3 with control chicks, which were statistically similar to each other. No significant differences were observed in heart and cecal relative weights between groups.

Table (2) Effect of ZONPs supplementationon body weight of broiler chicks.

Groups

3 day

week 1

week 2

week 3

week 4

week 5

*97.09^a

±1.14

261.13^b

±6.36

473.22^c

±11.38

907.22^b

±20.91

1446.04^b

±37.27

2004.26^b

±63.74

97^a

±1.21

290.65^a

±4.19

687.26^a

±12.88

1189.87^a

±16.49

1763.52^a

±28.92

2301.65^a

±34.19

96.91^a

±0.99

266.48^b

±6.68

625.83^b

±13.5

1144.09^a

±20.17

1674.35^a

±30.43

2189.17^a

±40.57

^{* Values are means ± SEM}

^{a,b,c, …Means in the same column with different superscripts are significantly different (P < 0.05)}

Table (3) Effect of ZONPs supplementation on feed consumption (FC), cumulative feed conversion ratio (FCR) and performance index (PI) of broiler chicks.

Groups	Weekly feed consumption (FC) (g / chick)					Cumulative FC (kg)	Cumulative FCR	Performance index (PI)
Groups	Week 1	Week 2	Week 3	Week 4	Week 5	Cumulative FC (kg)	Cumulative FCR	Performance index (PI)
T1	255	334.57	670.30	890.44	1106.2	3.257	1.625	123.34
T2	260.74	528.70	738.57	903.435	1105.5	3.537	1.537	149.75
T3	241.48	504.65	673.44	924.30	1093.1	3.437	1.570	139.437

Table (4) Effects of ZONPs supplementation on carcass and internal organs’ relative weight (gm / 100gm) of broiler chicks.

Groups	Carcass	Liver	Provent	Kidney	Heart	Intestine Wt	Cecal	Gizzard
T1	*68.29^b ±1.45	1.931^b±0.125	0.403^a ±0.01	0.639^a ±0.025	0.427^a ±0.014	6.648^a ±0.231	0.831^a±0.063	2.249^a ±0.147
T2	76.029^a ±0.72	2.152^b±0.046	0.348^b ±0.013	0.532^b ±0.012	0.403^a ±0.016	4.456^c ±0.275	0.802^a±0.036	2.018^b ±0.144
T3	72.285^a ±1.07	2.396^a±0.011	0.36^ab ±0.022	0.546^b ±0.032	0.439^a ±0.021	5.413^b ±0.296	0.806^a±0.018	2.276^a ±0.102

^{* Values are means ± SEM}

^{a,b,c, …Means in the same column with different superscripts are significantly different (P < 0.05)}

Blood biochemistry parameters

Table (5) lists the influences of dietary ZONPs treatments as feed supplement on serum biochemical analysis of broiler chicks. Data of the present research indicated that dietary inclusionof ZONPs at two both levels examined into broiler diets significantly decreases (P<0.05) serum concentrations of cholesterol (Chol), triglycerides (TG), low density lipoprotein (LDL) and uric acid and increased high density lipoprotein (HDL) value in comparison to control group. As well as, the highest concentration of serum enzymatic activity (ALT and AST) were observed in ZONPs- treated chicks groups (T2 and T3), but was significant only with increase the inclusion level of ZONPs at 60 mg (T3) as compared with controls. On the other hand, dietary ZONPs particle size had insignificant effects on serum glucose and creatinine concentration as their values were comparable among the groups.

Zinc content (ppm) in serum and body tissues (liver, breast and thigh muscle)

There were significant differences (P<0.05) among the treatments in the Zn content of liver, breast, thigh, and blood serum (Table 6).It could be noticed thatsupplementation of nano form of Zn into broiler diets at both two experimental level increased concentration of Zn inserum, breast, thigh muscles and liver as compared to those of control birds and these increase was dose dependent.

Table (5) Effects of ZONPs supplementation on serum biochemical parameters of broiler chicks.

Groups

Chol

(mg/dl)

HDL

(mg/dl)

LDL

(mg/dl)

Glucose

(mg/dl)

Uric

(mg/dl)

Creat

(mg/dl)

ALT

(U/L)

AST

(U/L)

*190.850^a

±3.46

59.771^b

±1.02

146.187^a

±1.99

94.509^a

±2.31

118.892^a

±2.92

6.786^a

±0.32

0.643^a

±0.03

36.863^b

±1.52

26.70^b

±1.07

172.496^b

±1.94

63.120^a

±2.25

139.046^b

±2.29

83.088^b

±1.34

118.438^a

±1.71

6.213^a

±0.27

0.594^a

±0.03

38.205^b

±1.22

27.241^b

±1.58

177.230^b

±2.1

65.0972^a

±1.63

141.892^ab

±1.65

81.890^b

±2.61

117.960^a

±2.26

5.991^b

±0.27

0.578^a

±0.02

42.061^a

±1.43

30.617^a

±1.29

^{* Values are means ± SEM}

^{a,b,c, …Means in the same column with different superscripts are significantly different (P < 0.05).}

Table (6) Effects of ZONPs supplementation on tissue and serum Zn concentration (ppm) of broiler chicks.

(liver)

(thigh)

(breast)

(serum)

Groups

3.736^b

±0.15

1.976^b

±0.11

0.923^b

±0.07

*1.107^c

±0.11

4.393^ab

±0.32

2.36^ab

±0.22

1.553^a

±0.18

1.563^b

±0.06

5.03^a

±0.21

2.613^a

±0.12

1.707^a

±0.11

2.017^a

±0.09

^{* Values are means ± SEM}

^{a,b,c, …Means in the same column with different superscripts are significantly different (P < 0.05)}

Histological observations:

Prominent and significant histological changes related to dietary treatments employed in the present study were demonstrated in the liver, kidney and intestinal tissues.

A- intestine:

Photomicrographs presenting the effect of ZONPs supplement on the intestinal villi (Vi) and intestinal crypt / gland (IG) of tested broilers. Histological examination for the sections of intestine of broiler chicks fed a supplemented diet with 40 mg /kg ZONPs in the present study are shown in figure (1A). The results showed obvious desquamation of the intestinal villi, profound increase in cell production of intestinal villi, goblet cells, intestinal crypt depth, and aggregated lymphocytes.

At the same time, histological study for the sections of intestine from the birds group fed supplemented diet with 60 mg ZONPs, exhibited marked degeneration, apoptosis of intestinal villi, and atrophy of intestinal crypt and no changes recorded in musculature layer (Fig. 1B).On the other hand, examined intestine of broiler chicks of control group presenting typical microvilli lined by enterocytes bearing brush border membrane and intestinal crypts and intact muscular layer (Fig 1C)

Morphometric analysis of intestinal villi length and crypt depth confirmed a high statistical difference between T2 group (40 mg ZONPs) and other groups. In contrast, no statistical difference recorded between T1 (Control group), and T3 (60 mg ZONPs). (Table 7).

Table (7) Effects of ZONPs supplementation on intestinal villi length and crypt depth of broiler chicks.

Groups	Intestinal villi length	Intestinal crypt depth
T1	9.61^b ± 0.46	3.19^b ± 0.17
T2	19.41^a ± 0.6	8.37^a ± 0.59
T3	8.74^b ± 0.32	3.45^b± 0.32

^{* Values are means ± SEM}

^{a,b,c, …Means in the same column with different superscripts are significantly different (P < 0.05)}

B- Liver:-

Photomicrographs for the section of the liver displaying the effect of ZONPs supplement on the normal hepatic architecture of the tested broilers are presented in figure (2).

Histopathological finding in the sections of the livers of 40 mg ZONPs - fed broiler (T2) revealed loss of hepatic cords arrangement and aggregation of inflammatory cells encircled portal area (Fig 2A) Moreover, hepatocytes

were dispersed between fat droplets, von Kupffer cells, and dilated sinusoids (Fig. 2B).). Microscopical examination of the liver of chicks treated with 60 mg ZONPs (T3) in their diets exhibited normal architecture around area of central vein supported by hepatocytes and sinusoids (Fig.2C).Unlike portal area that highlighted with aggregated inflammatory cells enclosing portal vein, hepatic artery, and hepatic cords (Fig 2D) Also, parenchyma of liver tissue in this group revealed some normal hepatocytes and others in karyorrhexis form. Von Kupffer cells engulfed RBCs besides fat droplets and dilated sinusoids were also detected (Fig. 2E). on the other hand, histological examination in the sections of the liver of the control group (T1) in the present study revealed that no histopathological alteration or any appreciable changes were observed. The liver assembled a normal structure of central vein, portal area and hepatic cords (Fig 2F).

C- Kidney:

Photomicrographs of renal cortex represented the effect of ZONPs supplementation as following; 40 mg ZONPs group displayed congested blood vessels, extravasation of blood cells between renal tubules and surrounding glomerular corpuscle, inflammatory cells infiltration, and epithelial desquamation and exudate noticed inside some tubules (Fig. 3A). Conversely, 60 mg ZONPs group highlighted degeneration of renal tissue including tubular disorganization and apoptotic cells, extravasated RBCS throughout tissue, and congestion in blood vasculature (Fig. 3B). Approaching control group, normal renal cortex structure emphasized with glomerular corpuscle, proximal convoluted tubules, distal convoluted tubules, and collecting tubules (Fig. 3C).

Growth performance

The role of the micronutrients zinc in animal health is well documented [39]. This nutrient cannot be stored in the body ,therefore, regular dietary intake of Zn is required to meet the physiological needs and functionality requirements [25,40].

The findings of the present study confirm that supplementing basal diet with ZONPsto broiler chicksresulted ina significant increase in body weight, increased feed consumption and improved feed conversion ratio compared with control chicks. In this respect, present results are in close agreement with those reported by [41] revealed that supplementation of Nano Zno into broiler diet at 2.5 ppm recorded significantly higher body weight gain, feed intake, and better feed conversion ratio. Also, [42]found a significant improvement in body weight, weight gain, feed intake, and feed conversion ratio in birds fed diets supplemented with 0.2 or 0.3 g/kg of ZONPs. Similarly, present results are in a harmony with [43] investigate the effect of different levels (20, 40, 60, 80 and 100 mg /kg feed) of dietary Nano zinc oxide supplementation on growth performance, and economic efficiency of cobb broiler chicks for 6 week of age and found that birds fed 40 mg ZONPs recorded the highest LBW, TBWG and the best FCR value compared with the other treatments. Besides, the same group (40 mg ZONPs / kg diet) significantly improved performance index value ( PI value).Furthermore, [44] reported that broiler chicks fed with ZONPs at 90 mg / kg for 35 days of age, showed an improvement in body weight compared with the bulk zinc oxide-administered group at 3000 mg /kg diet. Moreover, [45] evaluate the growth performance and economic benefits of nano-zinc compared to inorganic and organic sources of zinc supplementation in Giriraja; dual purpose chickenand concluded that growth performance (body weight, FC and FCR) and production economics were better during all weeks in chicken fed ZONPs supplementation at 60 mg/kg as compared with chicks fed a supplemented diets with Zinc sulphate and Zinc methionine at (30 and 60 ppm) respectively.

In addition, several previous results in the literature confirmed thatZnO nanoparticles at 20 to 60 mg/kg of diet could be appropriate levels and a considerable source of Zn to enhance BWG and achieve a better FCR of broiler chickens [46-54] and this was matched with the present work and that 40 mg/kg Nano-ZnO is the optimal level in diets.

The performance improvement may be attributed to the role of ZONPs in increasing the intestinal absorptive capacity as it increases mucosal length, villi length and width, and crypt depth [54], in addition to the higher absorption efficiencies of Zn nanoparticles due to its unique physical characteristics of transport and uptake, resulting in improved Zn bioavailability [13,55].

The positive effect of ZONPs supplementation on growth performance through the current study are corroborated with earlier works reported and may be due the important role of Zn in the overall performance and physiological process of poultry, as it is the integral component of more than 300 enzyme systems known as metalloenzymes, which are involved in metabolism of energy, nucleic acids, lipid and protein [4,56-58]. Furthermore, zinc is a known essential microelement for the growth performance of broilers so that, its deficiency in animals is characterized by appetite reduction, decreased FI, circulating levels of growth hormone (GH), and decreased hepatic production of Insulin growth factor (IGF-I) and growth falling in broilers [8,49; 59-61].

It is also, suggested that, zinc oxide in broiler can increase the concentration of sucrase in small intestine and result in increased carbohydrate absorption [62]. Zinc, in the composition of nanoparticles, has antioxidant and anti-stress properties, affects the intestinal microbiome of broiler chickens [63,64].

Carcass and relative organ weights.

The finding in the present results showed that supplementation of broiler diets with ZONPs at 40 or 60 mg / kg affect the carcass weight and internal organ weights as a percentage of live body weight. increasing carcass and liver weight and decreasing kidney, proventriculus, gizzard and intestine weight was observed as compared with control.

These results can be corroborated with earlier work reported by[43]as the relative weight of proventriculus at 40 and 100 mg ZONPs /kg of diet were significantly lower and the relative weight of the intestine was decreased by lowering the ZONPs levels with significant differences at 40 and 20 mg/kg of diet compared with the control. Moreover,weight of small intestine was lower and the liver weight was significantly increased as percentage of LBW in birds fed diet supplementation with 60 or 90 mg ZONPs / kg than other treatments. An explanation for increasing liver weight may be due to positive effect of ZONPs on better digestion and absorption of nutrient in gastrointestinal tract and or probably to be higher bioavailability zinc in the form of nanoparticles. As well as, the mention reason caused that zinc retention was higher in liver of broiler after absorption and inters to portal of blood[33].

current results was also consistent with those reported by [46]asrelative weight of gizzard was significantly reduced and no significant effect on the relative weight of heart with ZONPs (10 and 30 ppm) and the edible yield values in 20 and 40 ppm of ZONPs group were significantly higher than that in control groups.

A non-significant reduction in relative weight of gizzard of broilers fed diet containing 100 or 200 ppm ZONPs was reported by [28].In another study[65] suggested that dietary ZONPs at 80 mg/kg had significantly improved carcass yield and increased the relative weight of the digestive organs of broilers.,In addition, present study also clarified that addition of ZONPs to broilers’ diet significantly increased the eviscerated and edible yield compared with control groups. This finding was consistent with the previous investigations reported that dietary ZONPs significantly increased dressing percentage, carcass yield, and carcass weight at concentrations from 40 to 90 ppm [29, 53, 66].

Blood biochemistry parameters

Blood biochemistry parameters are important markers of physiological status in the body. The blood parameters of the treated groups varied significantly in terms of Chol, HDL, TG, LDL, ALT, AST, uric acid and serum creatinine concentration. Present results are congruentwith work reported by [46] who clarified that dietary inclusion of ZONPs at a dose of 20 ppm resulted in a significant reduction in the serum TG and uric acid concentration, while serum HDL level was significantly higher as compared with control broiler group.Also,supplementation of growing Japanese quail diets with Nano zinc at levels of 0.2 and 0.3 g/kg diet significantly decreased total cholesterol and LDL levels and had the highest value for HDL in the serum as compared to control [42]. In similar direction, serum total cholesterol and TG decreased significantly and activities of liver enzymes (AST and ALT) remained unchanged in the serum of Japanese quail fed ZnO-NPs at 30 or 60 mg / kg compared to the control [67].

The same trend was found in laying hens [47] and in broilers [33,68] current results also came in accordance with [69] records a reduction in cholesterol, TG, creatinine, uric acid and increased HDL with the supplementation of ZONPs at level of 20 or 40 mg /kg diet.

Present results are in close agreement withreport by [49] indicated that there was no significant difference in glucose levels and significantly decreased serum total cholesterol, triglyceride and very low-density lipoprotein (VLDL) by dietary supplementation of ZONPs to broiler chicks. This result could be attributed to the ability of Zn to induce glucagon secretion and suppress insulin secretion [70]. Significant improvements were observed in the health status (low blood cholesterol level and high ALT) of the birds by supplementing NZn to broiler diets at 0.06 mg/kg [71,72].

The significant increase in serum HDL and reduced blood serum TG and cholesterol in the present study is parallel with two previous study by the same author [29,73] on laying hen and broiler respectively administrated ZONPs. This finding may be due to the improvement in calories and fat intake after zinc supplementation and the fact that zinc’s role in enzymes systems, in that zinc forms an integral part of several enzymes (metalloenzymes), which plays a vital role in fat metabolism responsible for lipid digestion and absorption [74-79].

The decrease in cholesterol may be due to that Zn prevent cholesterol from absorption in gastro intestinal tract [80] and may promote the growth and activity of lactic acid bacteria, which reduces the cholesterol level by producing enzymes disintegrating bile salts and making them unconjugated [81].

On the other hand, present results came in contradictions with the data obtained by [52] indicated that the supplementation broiler diet with Nano-ZnO had no significantly affects the ALT and AST activates and increased serum cholesterol level. Possible reason for these differences is probably related to using doses and time of animal exposed to the Nano zinc, as, it has been reported that, level above 50 mg/kg of ZONPs increase the plasma level of ALT and AST [82].

Tissue Zinc concentrations.

The ability of Nano minerals to pass through the small intestine and distribute in the body are much greater than inorganic and organic minerals [83]. The Zn concentration in tissues and serum is often used as an indicator of the Zn status and storage in the body of animals as well as the level of Zn consumed by birds because low values are considered an early symptom of zinc deficiency [84,85].

Present study show that there were significant differences among treatments in the Zn concentration of liver, serum, breast and thigh muscles. Supplementation of ZONPs at both two dose supplementation increased amount of Zn in serum and all tissues examined compared with the control group and these increase linearly with increasing dietary ZONPs levels. These resultscame in agreement withthosereported in broilers by [44,63,86] and in layers by [7, 55, 87-89] where, the highest levels of zinc were detected in the livers, serum and muscles of birds fed a supplemented ration with ZONPs when compared with control one.Also, in Pekin ducksby[90] reported that zinc accumulation in plasma, breast muscle and liver were linearly increased with increasing zinc supplementation.

Additionally, in Japanese quails by [67] observed thatinclusion of ZONPs in the diet of Japanese quails induceda significant increase in the zinc concentration in liver compared to the control. They added that liver tissue retained a higher zinc concentration and the highest dose of ZONPs (60 mg/kg diet) was exclusively able to induce a significant increase in the concentration of zinc in the serum of Japanese quail. Similar results were obtained in rabbit by [91] who indicated thatnZnO supplementation at 30 and 60 mg / kg, significantly increased hepatic and serum zinc concentrations, which partly supported better absorption of nZnO and subsequently the positive relationship between nZnO supplementation and growth performance of rabbits.

The liver is an organ in the body that processes blood and helps to remove unwanted substances. Several studies indicates that Zn in the form of nanoparticles is able to penetrate into the hepatic cells via blood or interstitial space and being the liver as the main organ of Zn metabolism [63,92]. In addition, another theory that supports accumulation of Zn in the liver may be the activity of metallothionein, a cysteine-rich protein with the ability to bind divalent cations and control the pool and turnover of the microelements [93]. In this respect, [94] reported that dietary Zn supplementation linearly increases plasma and liver Zn concentrations in broilers. It is proved that the liver is sensitive to Zn supplementation because this organ acts as a Zn reservoir in the body, thus, it is expected that the birds receiving treatments supplemented with Zn should have a higher content of this element in their liver [95].

Furthermore, in two studies reported by the same author [96,97] it was indicated that an increase in Zn supplementation increased Zn content in broiler organs, in which the highest amount was observed in liver, thigh muscle, and breast muscle, respectively They also postulated that compared with breast muscle, the thigh muscle responded positively to dietary Zn supplementation, which is consistent to present study.

In this respect, present results are in line with [98-100] as Zinc content in chicken thigh is known to be higher than in breast. The same results were also reported by[63],where the concentration of zinc varies in tissues and the reason for this difference is due to the morphology, biochemical and functional state of the tissues. Additionally, according to [101] illustrated that the increase in serum Zn levels is related to the higher absorption and lower interaction with other complexed minerals in the digestive tract increasing the bioavailability of Zn bound to amino acids/proteins, and therefore it is easily absorbed because of such structures.

Earlier results revealed that the retention of zinc in the body increased as the dosage level of nano zinc oxide increases. This may be due to nano form trace element enter the animal’s body through direct penetration. Therefore, its utilization rate will be much higher than that of the ordinary inorganic trace elements [102]. Similarly, [65] also observed increased bioavailability of zinc in broilers when supplemented zinc in nano form as compared to birds fed with inorganic zinc. Moreover, the results of [33 , 57] revealed that Zn deposition was significantly increased in breast muscle and liver with dietary addition and/or substitution of inorganic Zn with nano Zn.

Furthermore, supplementation of nano-Zn (82.5 mg/kg diet) increased the Zn concentration in the liver of broilers compared to 110 mg/kg of ZnSO4 as microparticles [103].

Histological Section

A- Intestine:

Small intestine, its mucosal barrier in particular plays an essential role in absorption and maintaining health, preventing tissue injury and in ensuring adequate provision of dietary nutrients to the whole body. The height of villus and its ratio with crypt depth are most widely used as good indicators of mucosal integrity and intestinal function that correlated with better gut health and greater absorption of available nutrients [104,105].

Findings of this study inspected the impact of ZONPs supplement on the intestinal villi and intestinal crypt of tested broilers. Histological examination for the sections of intestine of broiler chicks fed a supplemented diet with 40 mg /kg ZONPs in the present study exhibited obvious desquamation of the intestinal villi, profound increase in cell production of intestinal villi and intestinal crypt, and aggregated lymphocytes. This was in consistent with the data recorded by [29] observed that dietary replacement of 60 mg inorganic zinc oxide with 45 and 30 mg of nano zinc/kg diet significantly increased villi length and width, crypt depth and villi length/crypt depth ratio compared with broiler chicks group fed on the basal diet with inorganic zinc oxide supplementation, which indicate improvement of absorptive capacity of different nutrients and improved feed efficiency. Also, [106] reinforced the pervious results when chicks fed diets with supplemental ZONPs at 50 ppm.

In a more comprehensive experimentconducted by [107] reported that supplementation of ZNOPs at the dose rate of 40 mg is a considerable feed additive for poultry with beneficial effects on intestinal changes. They showed a significant increase in villus height (VH), villus surface area (VSA) and total goblet cell (GC) count and villus height: crypt depth ratio (VH:CD) was also high in all parts of small intestine of 40 ZONPs supplemented birds. They clarified that the possible explanation for higher villus height may be due to higher bioavailability of zinc nanoparticles, so maintaining epithelial barrier integrity and function. The higher goblet cells could be another reason for higher villus height in intestinal segment, as acidic mucin is resistant to bacterial degradation resulting in less cellular damage [108]

Moreover, crypt development is essential to increase cell renewal rate and maturation in the gut. The increase in crypt depth of chicken supplemented with different levels of nano zinc/kg might provide more surface area for nutrient absorption by increasing enterocyte proliferation and intestinal mucin secretion because mucin-producing goblet cells are present mainly in the crypts [109].

The crypt plays a key role in the continuous renewal of villi because of its stem cell population whom continuous division throughout life allows replacement of villus epithelial cells [110]. In this regard, [111] illustrated that the improved dimensions of villus may linked with increased absorptive surface and higher proliferation of crypt cells of the villus due to the supplementation of Zn. Also, it wasindicated that zinc repaired intestinal injury by reducing the apoptotic index of ileal epithelial cells, enhancing villus height and crypt depth[112].

It speculated that, upon oral exposure, the dose of the NPs is the chief feature controlling the intensity of tissue damage. As some doses could improve the performance parameters of broilers without significant fear on broilers health (The no-observed-adverse-effect level (NOAEL)) and other doses caused tissue damage besides affect broilers health [113]. This can clarify the results of this study in the sections of intestine from the birds group fed supplemented diet with 60 mg ZONPs, they suffered from marked tissue degeneration, apoptosis of intestinal villi, and atrophy of intestinal crypt.

B- Liver:

The liver has the critical job of maintaining the body’s metabolic homeostasis. This includes the processing of dietary amino acids, carbohydrates, lipids and vitamins, removal of microbes and toxins in splanchnic blood and route to the systemic circulation; synthesis of many plasma proteins; and detoxification and excretion into the bile of endogenous waste products and pollutants xenobiotics.

Present results were in line with the findings of [44] and [114] investigated the effect of high (900 mg/kg b.w.), intermediate (600 mg/kg b.w.) and low (300mg/kg b.w) concentrations of zinc on the histology of the liver of broiler chicks. Pronounced degenerative, apoptotic, and inflammatory changes found in all birds as compared to the controlled group. These resultssuggest that due to the more accumulation of zinc in the liver of the broiler chicks, the chicks possess more stress and severe histological changes in their liver because the liver is the important site for zinc accumulation. Also, [115] reported that the distinguished apoptosis could be progressed from intercellular stress generated by ZONPs. They were reported to induce also ap53 upregulation, dermal fibrosis, and increased cell cycle evolution.

Previous literature displayed that ZONPs could rise neutrophils numbers with attraction to some proteins such as immunoglobulin and lipoproteins [116]. In this respect [117] attributed these pathological changes and their toxic effects to their solubility, resulting in increased intracellular Zn2+. Moreover, nanoparticles are expected to rise the existence of inflammation in diverse organs, consequently leading to the spreads of the inflammatory reactions [118]. Watson et al. [119] added that the administration of ZONPs hindered Kupffer cell phagosomes motility and thus conduct the hepatic injury. In this respect, supplementation of zinc causes cirrhotic liver that leads to a decrease in collagen, fibrin and reticulin as reported by [120].

C- Kidney:

Findings in this study monitored the outcomes of ZONPs supplementation on the normal renal tissue of the examined broilers. Approaching control group, normal renal cortex structure emphasized with glomerular corpuscle, proximal convoluted tubules, distal convoluted tubules, and collecting tubules. 40 mg ZONPs broilers group displayed congested blood vessels, extravasation of blood cells between renal tubules and surrounding glomerular corpuscle, inflammatory cells infiltration, and epithelial desquamation and exudate noticed inside some tubules. Conversely, 60 mg ZONPs group highlighted degeneration of renal tissue including tubular disorganization and apoptotic cells, extravasated RBCS throughout tissue, and congestion in blood vasculature. This agreed with [121] who revealed that zinc oxide nanoparticles exerted a destructive effect on the kidney. Moreover, it was found that these nanoparticles had a toxic effect on the renal tissue at a high concentration. Proximal tube imperfections, RBC crowding, and leukocyte leak specify the damaging properties of ZONPs in low doses on renal tissue.

Furthermore,the results of [122] explained that the kidney tissue of animals treated with ZONPs exhibited apoptosis along with necrosis, the reduced glomerular diameters induced by ZONPs may be a result of enhancing cell death which leads to kidney weight loss.In the study of [123] attributed the DNA damage and cytotoxicity occurred in the kidney cells was due to oxidative stress induced by ZONPs by decreasing SOD and GPx enzymes activity and increasing MDA contents. Consequently, oxidative stress can stimulate apoptotic or necrotic cell death pathways [124].

Zinc is widely used in the poultry industry for the growth of chicks, but in the limited amount. If we use zinc in higher than normal amount it affects the liver and other organs of the body. Therefore, it is necessary to take a dose of zinc, which helps to improve the proper growth of the chicks..

Generally, the obtained results confirmed that dietary supplementation of biologically synthesized nano sourced Zn at inclusion level of 40 or 60 mg/kg of diet have a significant positive effects in improvements of broiler performance, physiological status, carcass traits, serum biochemical parameters, lipid profile, intestinal health, serum and tissue concentration of Zn, and finally health status of broiler chicks. The improvements in studied criteria may be related to more bioavailable Zn resulted from the new properties of N-ZnO particles. current results showed also that the delivery of Zn in the forms of nanoparticles at concentration of 40 mg/kg to broiler diets was more efficient, beneficially and could be used as safe dose without any detrimental effect than high levels. Therefore, recommended inclusion of appropriate concentrations ZONPs, particularly 40 mg/kg to broiler diets to reach better results and that 40 mg/kg of ZONPs could be optimal supplementation level in diets.

Data Avialbilty: All data generated or analyzed during this study are included in this published articles.

Authors’contributions: Conceptualization, design, data collection, H.M.H; Formal analysis: H.M.H.; H M. S, Investigation, results and data discussion: H.M.H. R. E Methodology : H .M .H .;A.M. Abu taleb; H M. S; histological section:R E.Writing—original draft preparation: H.M.H, Validation and supervision:H.M.H. Zinc nanoparticles preparation and Synthesis.El R El. All authors read and approvedthe final manuscript

Funding: No Funding Applicable

Competing interests: The authors declare that they have no conflicts interest.

Ethics approval and consent to participate: the Animal care and experimental protocol were reviewed and approved by the Ethics Committee of local Experimental Animals Care Committee, Egyptian Nuclear Research Center

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Effects of Dietary Supplementation of Zinc Oxide Nanoparticles on Productive Performance, Physiological, Histological Changes and Tissues Zn Concentration in Broiler Chicks

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