Effect of Zinc Acetate on Broiler Nutrient Metabolism and Skeleton Characteristic

Liu Da-wei，Cao Shu-xin，Wei Jing-kun，Qin Wen-chao，Liu Yang，Li Jia-ning，and Xu Liang-mei

Institute of Animal Nutrition，Northeast Agricultural University，Harbin 150030，China

Abstract: The aim of this study was to determine the effects of supplementing organic forms of zinc (zinc acetate，ZnA) and inorganic zinc (zinc sulfate，ZnS) on performance，the gain to feed ratio (G : F) and skeletal characters in broilers from the first day to the 42th day. A total of 240-day-old broilers (Arbor Acres) were randomly distributed in four groups. A corn-soybean meal-based control diet containing recommended concentrations of inorganic trace minerals and other nutrients was prepared. On the basal diets，the control goup (CG) was supplemented with ZnS and the treatment group (TG) feeding diets were with different ZnA supplemental levels (80，100 and 150 mg · kg-1) of ZnA. Each group was allotted randomly to five replicates and 12 broilers in each replicate and fed ad libitum from the first day to the 42th day. The results showed that supplementation of ZnA significantly increased (p＜0.05)energy and phosphorus (P) compared to that fed ZnS on the 21th day. Calcium (Ca)，tibia weight，ash weight，tibia Ca and tibia P in TG (80 mg · kg-1 of ZnA) were the highest (p＜0.05). The pancreas Zn content of TG was higher than that in CG and increased with the supplementation of ZnA. On the 42th day，the content of energy，crude protein (CP)，Ca and tibia Ca in TG (80 mg · kg-1 of ZnA) were the highest (p＜0.05)，the change of pancreas Zn was the same as the results on the 21th day，but enter extract (EE)，tibia weight，tibia length，tibia width and ash weight were not affected (p＞0.05) by ZnA. The results of this study showed that ZnA was better than ZnS in improving the early growth performance，nutrient utilization rate in broilers，the length and weight of broiler tibia，it promoted the calcium，phosphorus deposition and normal growth of tibia，and the optimum dietary ZnA level to feed was 80 mg · kg-1. ZnA affected body Zn stores and the apparent metabolic rate of nutrients.

Key words: broiler，zinc acetate，nutrient metabolism，skeletal trait

Introduction

Zinc is an essential micro element with various biological activities and plays important roles in animals as well as human，especially in fast growing poultry(Liuet al.，2011). It is a constituent of metalloenzymes，such as carbonic anhydrase，carboxy peptidase and DNA polymerase (Salimet al.，2012). Zn is a cofactor of over 300 metalloenzymes and over 200 transcriptional factors (Mohammad-Rezaet al.，2004).In addition，deficiency of Zn deteriorates oxidative stress，due to metabolic and cellular damage by free radicals and reactive oxygen species (Salgueiroet al.，2000). Hence，Zn is typically supplemented to diets of commercial broilers to maintain optimum growth performance and health particularly，during early development (Shahet al.，2018; Bartlett and Smith，2003; Mwangiet al.，2017). In recent years，organic sources of trace minerals have been widely used in animal nutrition because of their higher bioavailability and potential for reducing environmental emissions than inorganic sources (Pierceet al.，2005).

Zinc deficiency increases production of free radicals and reactive oxygen species，which may result in an increase in oxidative damage to cell membranes，loss of appetite and reduced feed utilization which overall lead to growth retardation (Oteizaet al.，1996; Songet al.，2017). Many researchers have supplemented broiler diets with either inorganic Zn(Olukosiet al.，2018) or organic Zn (Aoet al.，2009;Aoet al.，2007) and observed an improvement in growth performance. However，others do not notice any effect of supplementation with either inorganic(Wanget al.，2002) or organic (Kiddet al.，1993) Zn on growth performance in broilers. Tissue content of Zn in broilers has linear relationship with dietary Zn level (Bartlett and Smith，2003). Leeson and Summers(2009) reported that there is adequate Zn concentration in corn-soybean diets for supporting optimal growth of broilers. It is possible that Zn requirements are variable for obtaining optimal growth rate (Liuet al.，2011)，immune system functions (Sunderet al.，2008;Levkutet al.，2018) and antioxidant enzyme activities.

The relative bioavailability of zinc acetate (compared to ZnSO4) in broilers is 161%，based on tibia Zn，and 248 % based on metallothionein mRNA expression (Richardset al.，2015). The contents of Ca and P in diets are increased，a common practice in diets of layers and pets，the relative bioavailability of chelated Zn is 441% (based on μg total tibia Zn).The difference in relative bioavailability might be attributed to the fact that inorganic zinc is generally ionized into zinc ions，while organic zinc is generally molecular，chemically stable，and has a slowrelease effect，thus，is more beneficial for biological absorption. However，published literatures on relative bioavailability of different sources of Zn are mixed.The inconsistencies are probably due to differences in the chemical characteristics (and thus bioavailability)among different organic Zn forms. Various forms of zinc (organic，chelate，complex，etc.) are used in experimental studies，as the complexity of the chemical structure increasing，the comparison among experimental results is more complicated.

Therefore，the objective of this study was to determine the effect of Zn supplementation of different forms (organic source: zinc acetate and inorganic source: zinc sulfate ) and levels of zinc acetate (ZnA)in broiler diets on apparent metabolic energy (AME)，tibia characteristics and economic returns.

Materials and Methods

Animals and management

A total of 240 1-day-old Arbor Acres (AA) male broilers were randomly distributed in four groups，each of which was replicated five times with 12 broilers per replicate，and was randomly allotted by body weight to mesh wire floored cages 61 cm×51 cm×36 cm (length，width and height，respectively). Each pen was provided with water and an individual feeder.All the broilers were allowed to consume mash feed and water at will. Room temperature was maintained at 32℃ for the first week and then decreased from 2℃ to 3℃ every week，until a temperature of 23℃to the end of the trial. The light-dark cycle was as the followings: from day 0 to day 7，there were 23 h of light and 1 h of darkness (lights off at 1 200 h and on at 1 000 h); from day 8 to day 42，there were 20 h of light and 4 h of darkness (lights off at 1 200 h and on at 1 600 h)(Leesonet al.，2010). All the experimental procedures complied with the Guidelines of Northeast Agricultural University Animal Care and Use.

Zn sources and diets

Zinc acetate (ZnA) and zinc sulfate (ZnS) used in the present work were provided by Shandong Grand Bio-Technology Co.，Ltd.，Heze，Shangdong，China. Zinc acetate (ZnA) with a purity of 99%，and Zn content was 29.6%. The purity of zinc sulfate (ZnS) was 98.6%，and Zn content was 35.2%.

The corn-soybean meal basal diets (Table 1) were formulated to meet or exceed nutritional requirements of broilers (NRC，1994) and NY/T33-2004 broiler nutritional standards in starter and grower stages，respectively. The crude protein (CP)，lysine，methionine，calcium，phosphorus and zinc of diets were determined by methods of AOAC International (2000). Dietary treatments included four groups，the control group was added to 40 mg · kg-1of ZnS (ZnSO4· H2O) and treatment groups were supplemented with 80，100 and 150 mg · kg-1of ZnA (Zn acetate) on the basal diets，respectively.

Table 1 Basal diet formula and nutrient in starter and grower stages (air-dry basis) (%)

Determination of nutrient apparent metabolic rate

On 21 and 42 days，two broilers of per replicate were randomly selected to measure AME，according to the national standard "Force-feeding Method of Determination of Chicken Feed Apparent Metabolic Energy Technical Regulations" (GB/T 26437-2010).The specific process was as the followings: fasting for 48 h，forced feeding of 60 g of diet，collection of excretions 48 h after feeding. Collected the excrement of every chicken and removed other things，such as feather and body debris，then weighed，and immediately dried at 65℃ until constant weight，room temperature regain 24 h.

AME (%)=(intake×feed energy-feces×feces energy)×100/intake

The energy was estimated using a Parr 6300 type oxygen bomb calorimeter (PARR，American). The crude fat contents were determined by ether extraction method; the crude protein contents were determined using Kjeldahl nitrogen determination method; Ca and P were determined by potassium permanganate method and colorimetric method，respectively.

Bone trait index

On 21 and 42 days，two broilers of per replicate were randomly selected，the broilers were slaughtered by severing the jugular veins and necropsied immediately.The pancreas，tibia，thigh muscle and breast muscle were rapidly excised and all the samples were stored at-20℃ until analyses.

Length，width and weighing of fresh weight of broiler tibia were measured using vernier caliper.Samples of tibia were dried at 105℃ for 12 h to constant weight. All the samples were then dry-ashed at 550℃ for 16 h，the ash weight and Ca and P contents of tibia were determined. At the same time，Zn content of pancreas was determined.

Determination of Ca and P in tibia: the ash was digested with 50% nitric acid，and the contents of calcium and phosphorus were determined by inductively coupled plasma-atomic emission spectrometry (ICPAES，ICP Modle，IRIS Advantage (HR)，Perkin Elmer Corporation，MA，USA); pancreatic Zn content determination method referred to Huanget al(2009).

Tibia ash content=(tibia bone ash weight/dry weight)×100%

Statistical analysis

Data were subjected to one-way ANOVA using the GLM procedure of SAS (SAS Institute，2004). Pen was the experimental unit. Orthogonal comparisons were applied for linear and quadratic responses of dependent variables to independent variables. In addition，nonlinear models (quadratic and asymptotic)were used to analyze the optimal dietary Zn level for broilers (Davieset al.，1979). Regression analyses were used to estimate Zn optimization (95% of the maximum or minimum response)，whenever a significant quadratic or asymptotic response (p＜0.05)was observed.

Results

Effect of ZnA on apparent metabolic rate of broiler nutrients

On the 21th day of the feeding trial，ZnA had no effect on CP and enter extract (EE) (p＞0.05). The energy，Ca and P were affected (p＜0.05) by dietary treatments.Between the control group (CG) and the treatment groups (TGs)，only energy and P had effects，but there were no effects (p＞0.05) in other aspects among different ZnA levels. At 80 mg · kg-1of zinc，the calcium metabolism of broiler chickens reached the maximum value. With the increase of zinc，the calcium metabolism showed a downward trend (Table 2); on the 42th day of the feeding trial，no difference was observed in EE. The changes in energy，CP and Ca were the same，the supplementation of 80 mg · kg-1of ZnA worked best. Compared to CG (40 mg · kg-1of ZnS) and TG (80 mg · kg-1of ZnA)，AMPs of P in 150 mg · kg-1of ZnA group were decreased by 3.32% and 4.51%，respectively (Table 2).

Table 2 Effect of ZnA on apparent metabolic rate of broiler nutrition

Effect of zinc acetate on skeletal traits of broilers

Dietary supplementation with different levels of ZnA did not influence the tibia length and tibia width (p＞0.05) of 21 and 42 days. The weight of the tibia and the sacral ash was affected (p＜0.05) by different levels of ZnA，and 80 mg · kg-1of ZnA supplementation worked best. On the 21th day，the weight of the tibia was maximal in 80 mg · kg-1of ZnA supplementation group compared with that in other treatment groups，but no effect was observed on the 42th day (p＜0.05).On the 21th day of the feeding trial，tibia Ca and P were decreased with increased ZnA supplementation，and 80 mg · kg-1of ZnA supplementation was the optimum dosage，supplementation with 80 mg · kg-1of ZnA in diets significantly increased the tibia Ca and P contents compared to those in the control diets(80 mg · kg-1of ZnS). But dietary supplementation of ZnA or ZnS did not change the tibia Ca and P contents on the 42th day of the feeding trial. On 21 and 42 days of the feeding trial，the supplementation of ZnA significantly increased pancreas Zn content as compared to the control group (supplementation with ZnS) (p＜0.05)，and pancreas Zn content was increased with increased ZnA supplementation (Table 3).

Economic benefit analysis

The feed cost of 80 mg · kg-1of ZnA supplementation was 0.001 Yuan more expensive than that of 40 mg · kg-1of ZnS supplementation，but economic benefits of 80 mg · kg-1of ZnA increased 1.56 Yuan per chicken by 7.9% compared with that of 40 mg · kg-1of ZnS; feed costs were increased by 0.001 Yuan with increased ZnA level，the benefit of the group of 100 mg · kg-1of ZnA and 150 mg · kg-1of ZnA was 0.58 Yuan and 0.61 Yuan higher than that in the control group of 40 mg · kg-1of ZnS per chicken，respectively.If a chicken farm rose 10 000 chickens，using 80 mg · kg-1of ZnA would be 15 600 Yuan more profitable than using 40 mg · kg-1of ZnS (Table 4).

Table 3 Effect of zinc acetate (ZnA) on pancreas Zn，skeletal traits and Ca and P contents of broilers

Table 4 Economic benefit analysis

Continued

Discussion

The present data were consistent with the hypothesis that organic zinc forms were more effective than inorganic zinc. Apparent metabolic rate of nutrients might directly reflect the utilization of feed in animals.Collectively，the data from this study indicated that an organic form of Zn [Zn(CH3COO)2] outperformed the inorganic ZnSO4· H2O in broilers. Similarly，Richardset al. (2015) reported that Zn (CH3COO)2was more bioavailable than ZnSO4，and the difference was greater when supplemented with higher levels of Ca and P in the diets. Burrellet al. (2004) reported that optimum body weight was achieved at 80 mg · kg-1supplementation zinc with a maize-soybean meal basal diet containing 30 mg · kg-1of Zn. It was found that the apparent metabolic rate of CP，energy and Ca of 80 mg · kg-1of ZnA were higher than that of the control group (40 mg · kg-1of ZnS) in broilers. Cousins(1979) reported that the dietary total nitrogen loss of chicken manure was significantly lower in zinc supplementation treatment than that in the control group，thus the metabolic efficiency of CP was increased. Hamidi and Pourreza (2009) and Liet al.(2015) added Zn methionine (organic) and Zn sulfate(inorganic) to the broiler diets，which intestinal structure was complete，the intestinal structure of the organic zinc group was complete，and the height of intestinal villus was better than that of the control group，duodenal crypt depth was significantly lower than that of the control group. Crypt became shallow suggested that the intestinal epithelial cell maturation rate was increased and the secretion function was enhanced，thereby affected digestion and absorption function (Ewtushiket al.，2000). Therefore，another reason for this results might be that 80 mg · kg-1of ZnA (organic) improved intestinal structure，increased villus height and crypt shallowness，and increased the number of mature epithelial cells，thus the digestion and absorption of feed were also increased.Zinc is a component and cofactor of many enzymes and hormones in animals，it also participates in the regulation of enzymes and hormones. Du and Zhao(2003) reported that the alkaline phosphatase and lactate dehydrogenase activities in serum of the laying hens were increased with the increase of Zn levels in diets. Alkaline phosphatase and lactate dehydrogenase are the key enzymes in glucose metabolism，and they also greatly influence amino acid metabolism. On the other hand，80 mg · kg-1of ZnA supplementation increased serum alkaline phosphatase and lactate dehydrogenase activities，promoted the metabolism of glucose and increased the synthesis of non-essential amino acids and the utilization of protein.

Bone is one of the zinc-rich tissues. The zinc bioavailability of the test substance was evaluated by the zinc content in tibia and pancreas. The content of zinc in tibia was considered to be the most sensitive criterion for assessing the bioavailability of znic in broilers，due to its susceptibility to the dietary zinc concentration (Wedekindet al.，1992; Wedekindet al.，1994). Lacking of zinc might cause slow growth of bones in broilers during growth. This experiment showed that compared with the control group，the length of tibia on 21 and 42 days was increased by 2.82% and 1.96% in 80 mg · kg-1of Zn acetate group，respectively. Skeleton mainly develops through entochondrostosis，and epiphyseal cartilage cell proliferation and differentiation are directly related to the backbone growth. The epiphyseal cartilage cells exist a kind of zinc transporter family，this protein has been confirmed that it is related to skeleton development. And previous studies showed that proliferation and differentiation of epiphyseal cartilage cells were inhibited by Zn deficiency. This experiment added 80 mg · kg-1of Zn acetate in diets，intracellular Zn of epiphyseal cartilage cell was sufficient，thereby promoted the zinc metabolism，proliferation and differentiation of chondrocytes and the length of the tibia. Ammermanet al. (1995) and Sandovalet al.(1997) reported that the tibia was the most sensitive biological tissue of trace elements zinc in broilers，and had been successfully used for bioavailability evaluation of zinc sources.

Compared with the control group，the tibia weight and oven dried-weight of broilers were significantly improved by 80 mg · kg-1of Zn acetate group; meanwhile，tibia calcium content was significantly higher than that of the control group on the 42th day. The previous studies showed that the content of zinc and calcium in the diets significantly affected tibia calcium content and bone calcification (Rossiet al.，2001; Swiatkiewicz and Koreleski，2008). Dietary znic deficiency significantly decreased serum osteocalcin and bone Ca compared to the normal zinc group. The result supported this experiment that 80 mg · kg-1of ZnA increased tibia Ca content. Additionally，80 mg · kg-1of ZnA supplementation increased apparent metabolic rate of calcium and phosphorus and promoted the tibial growth and development in broilers. In the bone，Ca and P exist in the form of hydroxyapatite crystal，P content is increased with the increased Ca content. Ca and P weights are about half the weight of the tibia，therefore，the increase of bone salt (Ca and P) deposition results in the increase of bone weight.

The pancreas is both an endocrine organ and an exocrine organ. It has a unique need for zinc in the production of digestive enzymes and biological processes，such as insulin packaging，secretion and signaling. Shannonet al. (2011) and Huang (2007)reported that the pancreas was the most sensitive soft tissue in response to dietary Zn concentration in broilers. In the present study，there was a significant increase (p＜0.05) of Zn source and levels on the pancreas Zn levels. Pancreatic Zn content of ZnA group was increased (p＜0.05) compared to ZnS group in broilers. This observation was explained by the fact that ZnA increased Zn mobilization from the liver back into circulation for maintaining Zn homeostasis，especially in pancreas，which had unique Zn requirement (Jouet al.，2009). In addition，the dietary supplementation of zinc acetate resulted in a higher (p＜0.05) Zn content in pancreas compared to zinc sulfate，and pancreatic Zn content was increased with increased zinc acetate levels. Henryet al. (1989)reported that the pancreas was the most sensitive soft tissue to the dietary Zn concentration in chicks from the first day to the 21th day. A dose-dependent response was observed between Zn supplementation and Zn content in tibia as well as in pancreas during the starter and grower periods，which was in accordance with previous researches (Williamset al.，1989; Staret al.，2012). In contrast to this findings，a corn-soybean meal diet supplemented with 0，30，60 and 90 mg · kg-1of organic Zn with different chelation strengths in broilers did not result in any significant difference in pancreas Zn concentration (Huanget al.，2009). These results suggested that the concentration of pancreatic zinc in broilers fed a diet supplemented with chelated zinc sources could not be used for the determination of zinc bioavailability.

Although ZnA is nearly 10 times more expensive than inorganic Zn (ZnS)，ZnA biological utilization is higher than ZnS. While Zn is a trace element，additive amount in feed is very little，therefore，it does not significant impacted the cost of feed. In this experiment，the economic benefit of 80 mg · kg-1of Zn acetate in broilers was the highest，and the economic benefit of each broiler was increased by 1.56 Yuan compared with that of the control group (40 mg · kg-1of Zn sulfate). According to this experiment，10 000 broilers were fed with zinc acetate，profit of 15 600 Yuan was obtained. The benefits were considerable.

Conclusions

In conclusion，compared with the addition of ZnS，ZnA was more beneficial to improve the early growth performance，nutrient utilization rate in broilers，the length and weight of broiler tibia，and to promote the calcium，phosphorus deposition and normal growth of tibia. In this test，the appropriate addition amount of ZnA was 80 mg · kg-1.