Comparative Effect of Zinc Concentration and Sources on Growth Performance, Accumulation in Tissues, Tibia Status, Mineral Excretion and Immunity of Broiler Chickens

25/December/2019 Approved: 27/February/2020 ABSTRACT This experiment was conducted to investigate the effect of feeding different concentrations and sources of zinc (Zn) on the growth performance, tissue mineral status, bone morphology and immunity responses in 0–4-week broiler chickens. Four hundred and forty 1-d-old broiler chickens were assigned randomly to 11 dietary treatments with 4 cages per treatment and 10 broiler chickens per cage in a completely randomized design. Dietary treatments were: corn–soybean meal basal diet (negative control), basal diet supplemented with 5 g yeast/kg (yeast), and basal diet supplemented with 20, 50, or 80 mg of added Zn/kg as ZnSO4, Zn-Met, or Zn-yeast in a 3 x 3 factorial arrangement of treatments. The results showed that broilers fed Zn supplemented diets had greater average weight gain and average feed intake than chickens fed the negative control diet ( p< 0.05). The Zn deposition in tibia, meat (thigh and breast) and excreta increased ( p< 0.01), regardless of source, in response to increasing dietary Zn concentrations. Zinc level increased dry weight of tibia bone and its large diameter. The strength of tibia bone as judged by Seedor index and breaking strength was improved ( p <0.01) with Zn concentration in increased diets. Furthermore, supplemental Zn up to 50 mg/kg improved immunity responses of broiler chickens ( p< 0.01). It is concluded that supplementation with 50 mg Zn may be sufficient for normal broiler growth up to 28 d of age and the dietary inclusion of organic Zn could be utilized more effectively when compared


INTRODUCTION
Zinc is an essential trace element that acts as a cofactor in many metabolic pathways including cell proliferation, growth, skeletal development, immune system, reproduction, hormone secretion, and antioxidant defense system, as well as many biochemical processes Swiatkiewicz et al., 2001;Ao et al., 2011;Muszyński et al., 2018). Thus, it is critical to use an optimal supplementation inclusion rate of Zn to allow poultry to reach their genetic potential and performance. The National Research Council (NRC 1994) recommended the minimum levels of 35 mg/kg in the diet that are necessary for optimum productivity for young broiler chickens. It has been reported that 71±13 mg Zn/g in a maize-soybean meal basal diet was necessary to maximize growth in broilers from hatch to 21 d of age (Huang et al., 2007). However, natural Zn concentrations in common feedstuffs are generally lower than the daily Zn requirement for poultry, leading to the necessity of dietary Zn supplementation. In practice, food manufacturers and producers formulate diets to contain 100-120 mg supplemental Zn/kg (Shyam Sunder et al., 2008;Feng et al., 2010). On the other hand high dietary Zn supplementation in diet may affect the balance of other trace elements (Abedini et al.,2017), and Azad SK, Shariatmadari F, Torshizi MAK, Chiba LI

Comparative Effect of Zinc Concentration and Sources on Growth Performance, Accumulation in Tissues, Tibia Status, Mineral Excretion and Immunity of Broiler Chickens
eRBCA-2019-1245 causes toxicity (Carl et al.,2003). High Zn consumption could also lead to increased excretion of Zn in feces, which causes environmental contamination (Pierce et al., 2005).
Research shows that the bioavailability of trace minerals in inorganic forms is low in poultry carcass (Cao et al., 2002). Therefore, enhancement of Zn bioavailability using more available sources can help to solve such problems. As such, organic mineral sources such as proteinate and amino acid chelates have been increasingly used in recent years because of their greater bioavailability and less excretion (Pierce et al., 2005). Previous studies have shown that the effect of different mineral sources, organic or inorganic, varies on production performance (Schlegel et al., 2013;Sahoo et al., 2014;Badawi et al., 2017). While in many studies, organically bound Zn has been demonstrated to have greater relative bioavailability than that of inorganic forms (Wedekind et al., 1992;Cao et al., 2000), others have seen no differences in bioavailability among organic and inorganic Zn sources (Hudson et al., 2004;Zakaria et al., 2017).
To our knowledge, the bioavailability of Zn-enriched yeast has not been previously fully elucidated and no comparative study (between this form and other organic and/or inorganic forms) has been carried out in broilers. Thus, the other objectives of the present study were to examine the effects of dietary supplementing different concentrations of Zn from various sources on growth performance, Zn excretion, leg development and immune responses in broiler chickens.

Birds, diets, and experimental design
The experimental protocol was approved by the animal-welfare committee of Tarbiat Modares University, and the animals were handled and treated in a humane manner.
Four hundred and forty 1-day-old broiler chickens (Ross 308) were assigned randomly to 1 dietary treatment group consisting of 4 replicates of 10 broilers in a completely randomized design arrangement of treatments. The house for the broilers was provided with programmed lighting and ventilation. Ambient temperature was gradually decreased from 32°C on day 1 to 22°C by the end of the experiment. Broilers were allowed ad libitum access to experimental diets and tap water containing no detectable Zn (<0.001 mg/L). Also, feed and water were provided using plastic instruments to minimize environmental Zn contamination.
The basal corn-soybean meal diets (Table 1), which was fed in mash form, were formulated to meet or exceed the NRC (1994) requirements for starter and grower broilers except for Zn. Dietary treatments included the un-supplemented basal diet with Zn (control), and supplemented with 20, 50, or 80 mg of Zn/kg as feed-grade Zn sulfate (ZnSO 4 •7H 2 O), Zn Methionine (Bioplex Zinc; Alltech, Inc ) and Zn-enriched yeast (that was produced on a laboratory scale as described by Kamran Azad et al 2014). All of the diets were calculated to contain equal concentrations of methionine. The background Zn concentration of the control diets were 24.4 and 21.6 mg/kg, in the starter and grower diets, respectively (Table 1). recorded at weekly intervals, starting from one day of age. Growth performance was evaluated in terms of average weight gain (AWG), average feed intake (AFI) and feed conversion ratio (FCR) at the end of each feeding period.

Sample collection and measurements
On d 28 of the experiment, 2 broilers from each replicate cage (8 broilers per treatment) were randomly selected within the cage following a 6-h fast, weighed individually, and were then killed.

Chemical analysis
Zinc concentrations in Zn sources, diets, tap water and tissues were determined by flame atomic absorption spectrophotometry (Model Avanta S, Atomic Absorption, GBC, Sydney, NSW, Australia) after wet digestions, as described by Sandoval et al. (1998). Briefly, samples of tissues and diets were dried at 105 ºC for 12 h. Tissues were predigested in HNO 3 , till charring was completed, then all samples were ashed dry at 550 ºC for 12 h, solubilized in HCl, and filtered through 42 Whatman paper.
The right tibia was removed and cleaned of the soft tissue before the bone was dried at 105 ºC for a minimum of 24 h. After 72 h-extraction in diethyl ether, the bone was dried for 12 h at 105 ºC and ashed at 550 ºC overnight in a muffle furnace (Yuan et al. 2011). Charred bones were digested as described before. The bone outer dimensions were measured by a digital caliper (Mitutoyo, Mizonokuchi Co, Utsonomiya, Japan). These bone parameters were measured on un-dried tibia samples. For determination of ash, the bones were placed in the oven for 24 h at 105 °C to dry completely and weighed. After that, the bones were ashed (650 °C for 14 h) and the ash weight was recorded. The material obtained from 2 duplicate was pooled.
For bone mechanical and geometric properties character, the following measurements were conducted. The Seedor index is the value obtained when the bone weight is divided by its length, as proposed by Seedor et al. (1991). It is used as a bone density indicator, the higher the value the denser the bone. Bone breaking strength was determined by material testing machine (Model H50KS; Hounsfield Co, London, England). The robusticity indexes were determined using the following formulas (Reisenfeld, 1972).
Robusticity index = bone length / cube root of bone weight To collect the excreta, 2 birds from each replicate were placed in a cage. A polythene sheet was attached under the cages of the birds. Feed and feathers were carefully removed. The excreta were homogeneously mixed replicate-wise, representative samples of excreta were collected in a moisture cup and oven-dried at 105°C for 24 hr, and finely ground for mineral analysis as described previously.

Immune response
On the 14th and 21th day of the experimental period, 2 birds were selected from each group and intramuscularly injected with 2 ml of 0.5% sheep red blood cell (SRBC). On the 21 st and 28 th day of the experiment, blood samples were collected and serum samples were used to measure humoral immunity. Antibody titre produced against haemagglutination was measured according to Peterson et al. (1999).

Statistical analysis
To test the effect of supplemented Zn, data were analysed using single degree of freedom contrast to compare all supplemental Zn treatment with the control treatment. Data were further analyzed by 2-way ANOVA (excluding control treatment) using the General Linear Model (GLM) procedure of SAS institute (SAS 2003). Replicate was considered as the experimental unit for all data. The model included main effects of supplemental Zn level, Zn source and their interaction. Influences regarding one (Zn level) of the main effects were based on irthohnal comparsion for linear response of dependent variables to independent variables. Duncan's multiple range tests was used to assess any significant differences at the probability level of p≤0.05 among the experimental treatments.

Growth performance
The broiler chickens fed diet without Zn supplementation had lower (p<0.01) AWG and AFI than those fed zinc supplemented diets (Table 2). Similarly, in the entire 4-week period, AWG increased with the dietary Zn content (p<0·05), up to dietary concentration of 50 mg Zn/kg. No additional response was observed at higher Zn concentrations. The inadequacy of Zn in the control diet depressed feed consumption (p<0.05); so that the lowest feed intakes were attributed to un-supplemented groups and the highest FI were shown in the 50 and 80 mg/kg of Zn. Moreover, the main effect of Zn level was not

Comparative Effect of Zinc Concentration and Sources on Growth Performance, Accumulation in Tissues, Tibia Status, Mineral Excretion and Immunity of Broiler Chickens
eRBCA-2019-1245 significant for feed conversion ratio (p<0.05). On the other hand the FCR was not affected with the dietary Zn sources but decreased with increasing Zn levels in both organic and inorganic groups.

Tibia Zn concentration
Zinc concentration in tibia was low in the broilers fed diets with no Zn supplementation (Table 3) but it increased in proportion to the dose of Zn supplementation to the basal diet and reached plateau at 50mg/kg (p<0.01). Tibia Zn concentrations were also strongly related to the Zn source origin, as organically bound Zn significantly increased the Zn content compared to inorganic supplementation (p<0.05). There were small changes in tibia Zn when broiler chickens were fed on either Zn-enriched yeast or commercial Zn methionine sources. The interaction between Zn level and its source was not significant for tibia Zn status.

Zinc content of meat and excretion
The Zn deposited in breast and thigh muscles reflected the level of dietary Zn (Table 3), the higher the inclusion level, the higher Zn content of muscles  (p<0.01). Zn accumulation was more substantial in birds fed organically bound Zn supplemented diets relative to those fed inorganic Zn counterpart (p<0.01).
Although the source of zinc affected the Zn content of thigh meat, it did not influence the Zn content of breast. Zinc excretion increased nearly three folds (p<0.01) from 175 ug/g (DM) control group to 515 ug/g (DM) for 80 mg/kg zinc level (p<0.01). There was no effect of zinc source on zinc excretion.

Mechanical and geometric character of bone
Mechanical and geometric characters of bones are presented in Table 4. Dry weight, Sidoor index, and breaking strength of tibia bone were affected by zinc content (p<0.05). However, there was no further increase in these parameters beyond 50 mg/ kg zinc levels. The zinc source had no significant effect on mechanical and geometric characters of bone measured. There was no interaction between level and zinc source effect on any parameters measured.

Immunity
The dietary treatment had no effect on weight of bursa Fabricius while the spleen weight was increased as compared to the control group (p<0.05). There was neither effect of zinc source nor interaction effect between zinc source and zinc level (p<0.05) on the weights of bursa Fabricius and spleen (Table  5). Although there was no significant difference in preliminary antibody titer among all birds, the chicks that had received zinc supplementation had a higher secondary antibody titer as compared to the control (p<0.05).

Growth performance
Zinc is an essential trace element for the normal function of numerous important structural proteins, enzymatic processes, lipid metabolism, hormone production and ultimately necessary for healthy growth and development of chicken. Inadequacy of Zn dosage in the bird diet reduces feed consumption and consequently body weight gain, but it could be reversed by Zn supplementation (Bao et al., 2007). Hudson et al. (2004) reported that different sources of Zn significantly affected body weight gain of broilers. Wedekind et al. (1992) studies indicated that the improvement in weight gain in broilers fed Znsupplemented diets may have resulted, in part, from increased consumption of basal diet, because anorexia Means in the same column with no superscript letters after them or with a common superscript letter following them are not significantly different (p<0.05).
1 There were 8 replicate consisting of samples taken from 2 birds per replicate (cage).

Comparative Effect of Zinc Concentration and Sources on Growth Performance, Accumulation in Tissues, Tibia Status, Mineral Excretion and Immunity of Broiler Chickens
eRBCA-2019-1245 is the most common symptom of Zn deficiency, which led to the depressed growth of broilers. The mechanisms involved in the effects of Zn deficiency on growth are unknown, but a reduction in food consumption may be a protective reaction to allow survival (MacDonald, 2000). An explanation for increased body weight gain may be due to the positive effects of Zn methionine on digestion and absorption of nutrients in the gastrointestinal tract and/or to a higher bioavailability of Zn in the form of Zn methionine. On the other hand, refer to the participation of Zn in protein and carbohydrate metabolism, the reduction in feed intake associated with a lack of Zn (that could reduce feed digestibility), and consequently impaired performance parameters in broilers (Bao et al., 2007). These results are in agreement with Huang et al. (2007) who found Zn supplementation induced increase in feed intake and weight gain.

Tibia Zn concentration
Zinc is the most abundant trace element in bone, being present at a concentration of up to 300 mg/kg (Grynpas et al., 1987), and has been considered an important factor in bone metabolism. Not surprisingly, adequate Zn concentration is required for growth, development and mineralization of bone (Bao et al., 2003). Huang et al. (2009) demonstrated that the Zn content of the tibia was significantly influenced by dietary levels. Previous studies reported that feeding birds diets with a Zn concentration greater than 85 mg/kg did not influence tibia Zn deposition (Wedekind et al., 1992). However, in this study, tibia Zn concentrations increased with Zn supplementation only up to 50 mg/kg and there was no change at 80 mg/kg. Huang et al. (2007) indicated that bone status is commonly used as an indicator of mineral adequacy in poultry diets.
Also in agreement with the present study, other studies indicated that different sources of Zn supplementation affected the levels of Zn in tibia (Ao et al., 2011;Idowu et al., 2011). Moreover, many research studies (Cao et al., 2000;Huang et al., 2009;Ao et al., 2011) indicated that the dietary supplementation of organic Zn can result in greater accumulation of Zn in tibia than the same supplemental concentration of inorganic Zn.
According to Loveridge (1992) bone is a complex heterogeneous tissue that supports the musculature, and thus its growth and development are intimately connected with overall body growth and thus making tibia Zn concentration a good predictor of whole-body growth. This study indicates that using 50 mg/kg Zn might meet the requirement for normal bone mineralization. In the present study, the same birds that obtained optimal body weight had highest tibia Zn. a a-b Means in the same column with no superscript letters after them or with a common superscript letter following them are not significantly different (p<0.05).
1 Mean values are based on data obtained from all (n=10) chicks from each of the 4 replicate pens per treatment (n=40 individual birds per treatment). Through analysis, a dummy variable was considered for the control and the three Zn sources so that the variable set to be zero for the birds not in the relevant group and one when they were in the relevant group. 1 antibody production based on log 10 Azad SK, Shariatmadari F, Torshizi MAK, Chiba LI

Comparative Effect of Zinc Concentration and Sources on Growth Performance, Accumulation in Tissues, Tibia Status, Mineral Excretion and Immunity of Broiler Chickens
eRBCA-2019-1245

Zinc content of meat and excretion
Many researchers indicated that dietary Zn level influence Zn content of nearly all types of tissues and organs (Park et al., 2004). The concept of "Functional foods", enriching poultry meat with different nutrients has attracted many researchers in recent decades (Peric et al., 2011). Our results though, indicate that Zn enrichment took place, the magnitude is not too high as compared to the control. The total Zn of thigh and breast meat in birds with no Zn addition is 26.4 (µg/g DM) while the highest level of Zn inclusion (80) resulted to total Zn content of 30 (µg/g DM).
On the other hand the magnitude of Zn excretion is nearly 2.5 folds; 176 (µg/g DM) for no zinc added diet as compared to 515 for 80 mg/kg added Zn. This imbalance between the ability to enrich a product and/ or cause industrial pollution is of great importance to consider. This proves that there is a limit for enriching meat with zinc (like most other minerals) beyond which is depleted through excretion (Keen et al., 2003).
However, Bao et al. (2007) had shown organic minerals decreases minerals of excreta. Salim et al. (2010) indicated that chelated minerals (such as organic zinc) could resist interferences from dietary anti nutritional factors in the digestive tract and directly reach the intestinal brush border, where it is hydrolyzed and absorbed as an ion into to the blood, resulting in a greater availability.

Mechanical and geometric character of bone
This study suggests that using 50 mg Zn/kg (from yeast Zn) might meet the requirement for normal bone mineralization. Although Zn supplementation at specific levels is essential to optimize bone breaking resistance, it has been reported that higher levels of Zn in the diet appear to interfere with the absorption and utilization of Ca and P, particularly above 80 ppm, and could decrease the bone mineralization (Underwood & Suttle 1981) which was also observed in our study.
Many previously conducted studies show the negative impact of Zn deficiency on bone growth, and disorders that are associated with reduced activity of the growth plate (Brown et al., 1978). Scrimgeour et al. (2007) indicated that when Zn is not supplied sufficiently, proliferation, differentiation and survival of the bone cells are compromised. It seems that Zn, by increasing the number and activity of osteoblasts, leads to the deposition of calcium in the diaphysis of bones and increase mineralization in the tibia. Masayoshi and Hidetoshi (1989) demonstrated that Zn enhanced the effects of vitamin D on bone metabolism by stimulating the synthesis of DNA in bone cells. On the other hand, Zn by stimulating bone metabolism and bone protein synthesis by increasing the activity of enzymes such as alkaline phosphatase, is involved in increasing bone mineralization and strength (Yamaguchi et al., 1988). It has also been suggested that Zn is involved in insulinlike growth factor I-(IGF-I) production that increases the collagen, DNA and bone matrix syntheses (Hock et al., 1988). Therefore, many effects of Zn on bone metabolism may be related to nucleic acid and protein metabolism.

Immunity
Since the spleen is the organ that is directly related to antibody production, it is expected that its weight be directly related to antibody production (Steiniger and Barth, 2000). While there are no concrete evidences as to the effect of zinc sources on weight of the brusa Fabricius and the spleen, there is not much dispute in regard to the effect of zinc levels on these organs. Yu et al. (2005) showed that diet lacking in zinc results to lower weight of the spleen. Increase of the weight of bursa (and not the spleen) in this study was similar with the result of Bartlett and Smith (2003), who showed a slight increase in the weight of lymphoid organs. Also, in their experiments on broilers reported that thymus, spleen, and bursa of Fabricius increased linearly with increasing dietary Zn (from 35mg/kg to 68mg/kg). These findings could be due to the role of Zn in the growth and function of lymphocytes.
The results of this study indicated that Zn supplementation improves immune responses, as compared to the control. The immune system is dependent on the functions of cellular metabolism. Zn is ubiquitous in cellular metabolism and functions both structurally and catalytically in metalloenzymes (Bartlett & Smith, 2003).
In this study the birds with higher spleen weight had higher SRBC secondary titer. However, the maximum secondary SRBC immune response was seen in birds receiving 50 mg/kg zinc (p<0.05) but there was no differences with the 80mg/kg level. In the other hand according to Sunder et al. (2008), humoral and cellmediated immune responses were significantly higher in broilers supplemented with 80 mg/kg or greater amounts of Zn than those supplemented with less than 80 mg/kg of Zn. Hudson et al. (2004) observed a higher cellular immune response to PHA and antibody titres against the Newcastle disease in broiler breeders fed with diets supplemented with organic sources of Zn, as compared to inorganic sources. Zinc is essential for thymulin, a thymic hormone that regulates T lymphocyte maturation. Thus, birds fed with diets supplemented with a more available Zn source might have more thymulin activity and, therefore, promote immune responses through the increased maturation of T-lymphocytes and activation of B-lymphocytes by T-helper cells.

CONCLUSION
This experiment was conducted to examine the effect of different levels of zinc from different sources on broiler chickens performances. The levels were chosen to reflect the earlier proposed zinc requirement (NRC), recommended levels to present faster grower birds, and higher level than commonly practiced. As many earlier experiments to determine zinc requirement were done with inorganic zinc, it was presumed that organic zinc being more bioavailable will enhance the broiler performances at lower levels as compared to inorganic Zn. The main objective of our study apart from general performances criteria was to evaluate the effect of zinc on tissue and excretion zinc level and tibia morphology. According to our results, the optimal dietary Zn requirements for 0-4-wk-old broilers were 50 mg/kg diet. Zinc level above 50 mg/kg not only did not improved performances criteria further, it adversely increased zinc excretion and ultimately environment pollution. Unlike many other studies, our results did not indicate that zinc sources (sulfate vs organic) within the range tested in this experiment had much influence on broiler performances.

DISCLOSURE STATEMENT
No potential conflict of interest was reported by authors.