Effects of Dietary Supplementation With Earthworm Powder on Production Performance, Blood Characteristics, and Heavy Metal Residues of Broiler Pullets

Effects of Dietary Supplementation With Earthworm Powder on Production Performance, Blood... SUMMARY Earthworms have been studied as a feed additive and animal protein source for poultry for many years. The interest in earthworm use for manure management has greatly increased over the past few decades. However, it is unknown whether earthworm powder in the diet poses a potential heavy metal risk for broiler pullets. This study was conducted to investigate the safety of adding earthworm powder in the diets of broiler pullets. A total of 240 Ningdu Yellow broiler pullets were randomly assigned to 4 groups receiving diets supplemented with 0%, 1%, 3%, or 5% earthworm powder for 30 d. Feed conversion ratios were improved by 12.64% and 22.45%, when 3% and 5% earthworm powder, respectively, were added (P = 0.02). Supplementing the diet with 5% earthworm powder had no negative effects on the growth of pullets, and increased antioxidant enzyme (superoxide dismutase, catalase, and glutathione peroxidase) activities in the liver (P < 0.05). The liver contained higher levels of heavy metals than did the thigh and chest muscles. All heavy metal residues in the thigh and chest muscles, as well as the liver, were below China's permissible limits. These results suggest that dietary supplementation with 1%–5% earthworm powder is safe for broiler pullets. DESCRIPTION OF PROBLEM The presence of animal protein in poultry feed is essential for the growth and health of the reared birds. Fish meal, because of its balanced amino acid contents, is the conventional source of animal protein in poultry feeds [1]. However, because of the increasing cost of high-quality fish meals and due to the declining stocks of fish obtained from capture fishery, at present, there is a need to search for alternative sources of animal protein for incorporation into poultry feeds [2]. Earthworm meal is an easily available resource that can be used as a feed additive and an animal protein source for poultry [3, 4]. Many scientists have reported that the rapid breeding and composting potential of earthworms may be effectively utilized as an economic animal feed protein for the fish and poultry industries worldwide [3, 4, 5]. Bahadori et al. [6] showed that earthworm meal is rich in lysine (4.44%), methionine (1.20%), and metabolizable energy (3,258 KcalME/kg). Jang Ho [7] observed that using earthworm meal to feed broiler chickens could improve feed intake, protein digestibility, and weight gain. Over the last few decades, as regulations for the disposal of animal manure have become more rigorous [8, 9], interest in the use of earthworms as an ecologically sound method of manure management has increased dramatically. Earthworms, many cultured on livestock manure, can take up and accumulate heavy metals, such as cadmium, mercury, and gold, in their tissues [10]. As such, even if the levels of heavy metals in poultry diets are maintained below the permitted levels based on the National Feed Hygiene Standard GB 13078–2001 of the People's Republic of China, addition of heavy metal-contaminated earthworm meals in the poultry diets may lead to the deposition of heavy metals in the poultry carcasses; these metals can then be transferred to humans via consumption of the contaminated meat. Contamination with heavy metals is a serious toxicity threat, since they are capable of bioaccumulation and biomagnification in the food chain [11]. These pollutants have direct toxic effects on the physiology because they are stored or incorporated into tissues, sometimes permanently [12, 13]. Ibáñez et al. [14] evaluated the toxicology of earthworm (Eisenia fetida) meals used as a protein source in rats. The continued development of intensive livestock production systems has led to an increased heavy metal contamination of livestock manures, thereby increasing the importance of evaluating the heavy metal contents in the broiler chickens fed earthworm meal. Earthworms have been widely used in traditional medicine for thousands of years, as they possess rich natural sources of antioxidants [15]. Supplementation of diets with earthworm meal powder (henceforth earthworm powder) might improve the antioxidant system of broilers, which may protect them from the oxidative damage inflicted by excess reactive oxygen species that otherwise promote cell injury and death in a broad variety of disorders, and are considered to cause important pathophysiological conditions [16]. Thus, it is necessary to investigate the possible effects of earthworm powder used as a feed additive on some antiperoxidative enzymes present in the blood serum of the broilers. The following experiments were conducted to evaluate the heavy metal concentrations and antioxidants in the blood serum resulting from the addition of earthworm powder to the diets of broiler pullets. Specifically, our objectives were to evaluate the effects of earthworm powder on production performance, blood characteristics, and heavy metal contents of the broiler pullet carcass. MATERIALS AND METHODS Preparation of Earthworm Powder Earthworm powder was prepared following the method of Ibáñez et al. [14]. The cultivated earthworms used in this experiment were grown in a pig manure–wheat straw mixture [17]. Moisture, crude protein, and metabolic energy of the powder were determined by the usual feed procedures [18], and crude fat by the Association of Official Analytical Chemists method [19]. Amino acid composition was determined with an amino acid auto analyzer [20]. The powder was analyzed for total heavy metals (Zn, Cu, Pb, Cd, As, and Cr) by aqua regia digestion and analysis using inductively coupled plasma spectrophotometry [21] or graphite furnace atomic absorption spectrophotometry (GFAAS). Experimental Design The care of laboratory animals and the experimental methods involving animals were approved by Jiangxi Agricultural University Institutional Animal Care and Use Committee. A total of 240, 9-wk-old Ningdu Yellow broiler pullets were randomly assigned to one of 4 treatments in an experiment that lasted 30 d. There were 5 replicates (12 pullets per replicate) for each treatment. Pullets were weighed individually at the onset of the experiment and then assigned to treatments in a randomized block design. Pullets were provided with free access to water through the nipple of an automatic drinker and feed through a common trough feeder. The composition of the corn and soybean meal basal diet is shown in Table 1, and the heavy metal contents in the diets were below China's permissible limits (GB13078–2001). Dietary treatments consisted of the corn and soybean meal basal diet supplemented with 0% (control EP 0), 1% (EP 1), 3% (EP 2), or 5% earthworm powder (EP3; Table 1). To balance the protein content of the diet, the proportion of soybean was decreased with the increasing earthworm powder content. Because earthworm meal is rich in lysine and digestible energy (DE), as evident from the estimated nutrient proportions of the earthworm powder (Table 2), the nutrient level of threonine and DE in the diet also increased in correspondence with the supplementation of earthworm powder (Table 1). The experimental diets were formulated in accordance with standard chicken breed recommendations (NY/T33-2004). Before the beginning of the experiment, 9-wk-old hens were purchased and provided with the basal diet for a 7-wk adjustment period. Feed intake and body weight were recorded at the end of the experiment (30 d). Table 1. Composition of diet. Groups Items EP0 EP1 EP2 EP3 Diet composition (%) Corn 68.20 68.20 68.20 68.20 Soybean bean 21.30 20.16 17.87 15.58 Wheat bran 4.00 4.14 4.43 4.72 Earthworm powder 0 1 3 5 Limestone 1.10 1.10 1.10 1.10 Dicalcium phosphate 1.40 1.40 1.40 1.40 Oili 2.70 2.70 2.70 2.70 Sault 0.30 0.30 0.30 0.30 1% Premix 1 1 1 1 Nutrient level DE (MJ/kg) 12.98 13.38 14.16 14.95 Crude protein (%) 16.00 16.00 16.00 16.00 Met + Cys (%) 0.69 0.69 0.69 0.70 Lys (%) 0.79 0.80 0.82 0.83 Thr (%) 0.70 0.71 0.72 0.73 Calcium (%) 0.82 0.82 0.81 0.81 Total P (%) 0.59 0.58 0.57 0.56 Available P (%) 0.30 0.29 0.29 0.29 Cu (mg/kg) 9.26 7.23 13.59 13.41 Zn (mg/kg) 79.08 104.39 102.47 105.46 Cr (mg/kg) 0.25 0.45 1.07 1.84 Cd (mg/kg) 0.03 0.04 0.08 0.07 As (mg/kg) 3.97 3.76 5.80 3.68 Pb(mg/kg) 1.17 1.31 1.67 1.77 Groups Items EP0 EP1 EP2 EP3 Diet composition (%) Corn 68.20 68.20 68.20 68.20 Soybean bean 21.30 20.16 17.87 15.58 Wheat bran 4.00 4.14 4.43 4.72 Earthworm powder 0 1 3 5 Limestone 1.10 1.10 1.10 1.10 Dicalcium phosphate 1.40 1.40 1.40 1.40 Oili 2.70 2.70 2.70 2.70 Sault 0.30 0.30 0.30 0.30 1% Premix 1 1 1 1 Nutrient level DE (MJ/kg) 12.98 13.38 14.16 14.95 Crude protein (%) 16.00 16.00 16.00 16.00 Met + Cys (%) 0.69 0.69 0.69 0.70 Lys (%) 0.79 0.80 0.82 0.83 Thr (%) 0.70 0.71 0.72 0.73 Calcium (%) 0.82 0.82 0.81 0.81 Total P (%) 0.59 0.58 0.57 0.56 Available P (%) 0.30 0.29 0.29 0.29 Cu (mg/kg) 9.26 7.23 13.59 13.41 Zn (mg/kg) 79.08 104.39 102.47 105.46 Cr (mg/kg) 0.25 0.45 1.07 1.84 Cd (mg/kg) 0.03 0.04 0.08 0.07 As (mg/kg) 3.97 3.76 5.80 3.68 Pb(mg/kg) 1.17 1.31 1.67 1.77 Note: The premix fodder contains: methionine, 1.0 g/kg; lysine, 0.7 g/kg; threonine, 0.8 g/kg; choline chloride, 0.5 g/kg; VA, 19,000 IU/kg; VD3, 5200 IU/kg; VE, 38 IU/kg; VB12, 0.022 mg/kg; VB6, 5.6 mg/kg; VK3, 3.8 mg/kg; riboflavin, 11.4 mg/kg; VB1, 8 mg/kg; D-pantothenic acid, 22.8 mg/kg; nicotinic acid, 38 mg/kg; biotin, 0.15 mg/kg; folic acid, 0.56 mg/kg; Mn, 85 mg/kg; Cu, 10 mg/kg; iodine, 0.35 mg/kg. View Large Table 1. Composition of diet. Groups Items EP0 EP1 EP2 EP3 Diet composition (%) Corn 68.20 68.20 68.20 68.20 Soybean bean 21.30 20.16 17.87 15.58 Wheat bran 4.00 4.14 4.43 4.72 Earthworm powder 0 1 3 5 Limestone 1.10 1.10 1.10 1.10 Dicalcium phosphate 1.40 1.40 1.40 1.40 Oili 2.70 2.70 2.70 2.70 Sault 0.30 0.30 0.30 0.30 1% Premix 1 1 1 1 Nutrient level DE (MJ/kg) 12.98 13.38 14.16 14.95 Crude protein (%) 16.00 16.00 16.00 16.00 Met + Cys (%) 0.69 0.69 0.69 0.70 Lys (%) 0.79 0.80 0.82 0.83 Thr (%) 0.70 0.71 0.72 0.73 Calcium (%) 0.82 0.82 0.81 0.81 Total P (%) 0.59 0.58 0.57 0.56 Available P (%) 0.30 0.29 0.29 0.29 Cu (mg/kg) 9.26 7.23 13.59 13.41 Zn (mg/kg) 79.08 104.39 102.47 105.46 Cr (mg/kg) 0.25 0.45 1.07 1.84 Cd (mg/kg) 0.03 0.04 0.08 0.07 As (mg/kg) 3.97 3.76 5.80 3.68 Pb(mg/kg) 1.17 1.31 1.67 1.77 Groups Items EP0 EP1 EP2 EP3 Diet composition (%) Corn 68.20 68.20 68.20 68.20 Soybean bean 21.30 20.16 17.87 15.58 Wheat bran 4.00 4.14 4.43 4.72 Earthworm powder 0 1 3 5 Limestone 1.10 1.10 1.10 1.10 Dicalcium phosphate 1.40 1.40 1.40 1.40 Oili 2.70 2.70 2.70 2.70 Sault 0.30 0.30 0.30 0.30 1% Premix 1 1 1 1 Nutrient level DE (MJ/kg) 12.98 13.38 14.16 14.95 Crude protein (%) 16.00 16.00 16.00 16.00 Met + Cys (%) 0.69 0.69 0.69 0.70 Lys (%) 0.79 0.80 0.82 0.83 Thr (%) 0.70 0.71 0.72 0.73 Calcium (%) 0.82 0.82 0.81 0.81 Total P (%) 0.59 0.58 0.57 0.56 Available P (%) 0.30 0.29 0.29 0.29 Cu (mg/kg) 9.26 7.23 13.59 13.41 Zn (mg/kg) 79.08 104.39 102.47 105.46 Cr (mg/kg) 0.25 0.45 1.07 1.84 Cd (mg/kg) 0.03 0.04 0.08 0.07 As (mg/kg) 3.97 3.76 5.80 3.68 Pb(mg/kg) 1.17 1.31 1.67 1.77 Note: The premix fodder contains: methionine, 1.0 g/kg; lysine, 0.7 g/kg; threonine, 0.8 g/kg; choline chloride, 0.5 g/kg; VA, 19,000 IU/kg; VD3, 5200 IU/kg; VE, 38 IU/kg; VB12, 0.022 mg/kg; VB6, 5.6 mg/kg; VK3, 3.8 mg/kg; riboflavin, 11.4 mg/kg; VB1, 8 mg/kg; D-pantothenic acid, 22.8 mg/kg; nicotinic acid, 38 mg/kg; biotin, 0.15 mg/kg; folic acid, 0.56 mg/kg; Mn, 85 mg/kg; Cu, 10 mg/kg; iodine, 0.35 mg/kg. View Large Table 2. Nutrient Ingredient of Earthworm Powder. Composition Earthworm powder Crude protein (%) 57.85 Crude fat (%) 3.50 Total amino acids (%) 50.80 Total essential amino acids (%) 38.31 Lys (%) 4.04 Thr (%) 2.64 DE (MJ/kg) 12.46 Composition Earthworm powder Crude protein (%) 57.85 Crude fat (%) 3.50 Total amino acids (%) 50.80 Total essential amino acids (%) 38.31 Lys (%) 4.04 Thr (%) 2.64 DE (MJ/kg) 12.46 View Large Table 2. Nutrient Ingredient of Earthworm Powder. Composition Earthworm powder Crude protein (%) 57.85 Crude fat (%) 3.50 Total amino acids (%) 50.80 Total essential amino acids (%) 38.31 Lys (%) 4.04 Thr (%) 2.64 DE (MJ/kg) 12.46 Composition Earthworm powder Crude protein (%) 57.85 Crude fat (%) 3.50 Total amino acids (%) 50.80 Total essential amino acids (%) 38.31 Lys (%) 4.04 Thr (%) 2.64 DE (MJ/kg) 12.46 View Large Sampling and Measurements A total of 10 birds were selected at random from each treatment on the initial day of the experiment. Blood samples were collected from the wing vein of each pullet on the initial and final days of the experiment, using a sterilized syringe and both nonheparinized tubes and K3EDTA vacuum tubes to obtain serum and whole blood, respectively. The blood samples were then centrifuged at 2000 × g at 4°C for 20 min within 1 h of collection to separate the serum. The total protein (TP), albumin, glutamic oxaloacetic transaminase (GOT), glutathione peroxidase (GSH-Px), alkaline phosphatase (ALKP), superoxide dismutase (SOD), catalase (CAT), alanine aminotransferase (ALT), and cholinesterase (CHE) were measured using commercial laboratory kits [22] with a spectrophotometer [23]. On the final day of the experiment, all the pullets were killed by decapitation, and samples (thigh muscles, chest muscles, and liver) were collected in polyethylene bags, stored at –18°C, and analyzed as soon as possible. The heavy metal contents in the homogenated tissues were estimated using inductively coupled plasma spectrophotometry or GFAAS. Statistical Analysis Analysis of variance was performed to analyze the data using IBM SPSS 19.0 [24]. Tukey's HSD was used in the analysis for multiple comparisons. The significance level was set at P < 0.05. RESULTS AND DISCUSSION Production Performance Table 3 summarizes the effects of dietary earthworm powder supplementation treatments on the enhancement of body weight, feed intake, and feed conversion ratio in 9-wk-old pullets. The measurements of all these parameters increased with the increasing proportion of the earthworm powder added. The feed conversion ratio was improved by 12.64% and 22.45% when 3% and 5% earthworm powder, respectively, were added (P = 0.02). Earthworm meal is equal to or surpasses other feed supplements, like fish meal and meat meal, as an important animal protein source for poultry. Table 1 showed that the nutrient level of threonine and DE in the diet increased in correspondence with the supplementation of earthworm powder. These results were in accordance with the findings of Bahadori et al. [6]. They showed that earthworm meal is rich in lysine and metabolizable energy, which may be the cause of improved feed conversion. Tuan [25] indicated that fish fed with diets containing earthworm powder had similar or higher growth rates, protein efficiency, and energy retention than that of a control group fishes. Prayogi [26] reported that weight gain and the feed-conversion ratio improved in quails fed diets supplemented with 10% earthworm meal. Taboga [27] indicated that the amino acid composition of earthworms was close to the amino acid requirements of chickens. In addition, Julendra et al. [28] indicted that the improvement in the body weight gain in broilers fed earthworm meal may have been caused by the antibacterial characteristics of the earthworm's coelomic fluid, which had the potential to thin the intestinal mucosa and increase nutrient absorption and feed efficiency [3]. Table 3. Growth Performance of broiler pullets fed earthworm powder. Parameters EP0 EP1 EP2 EP3 P-value Initial weight (g) 729.67 ± 1.34 731.33 ± 1.67 730.33 ± 2.67 730.33 ± 1.75 0.53 Final weight (g) 1073.44 ± 13.79a 1050.02 ± 5.69a 1106.67 ± 13.78b 1142.86 ± 18.07c 0.01 Daily gain (g) 11.46 ± 0.22a 10.62 ± 0.18a 12.54 ± 0.17b 13.75 ± 0.18c 0.02 Feed conversion ratio 5.30a 5.62a 4.63b 4.11c 0.02 Parameters EP0 EP1 EP2 EP3 P-value Initial weight (g) 729.67 ± 1.34 731.33 ± 1.67 730.33 ± 2.67 730.33 ± 1.75 0.53 Final weight (g) 1073.44 ± 13.79a 1050.02 ± 5.69a 1106.67 ± 13.78b 1142.86 ± 18.07c 0.01 Daily gain (g) 11.46 ± 0.22a 10.62 ± 0.18a 12.54 ± 0.17b 13.75 ± 0.18c 0.02 Feed conversion ratio 5.30a 5.62a 4.63b 4.11c 0.02 Note: values are mean ± SE in each group. Values not sharing a common superscript differ significantly at P < 0.05. EP0, EP1, EP2, EP3 indicates diets supplemented with 0, 1%, 3%, or 5% earthworm powder, respectively. View Large Table 3. Growth Performance of broiler pullets fed earthworm powder. Parameters EP0 EP1 EP2 EP3 P-value Initial weight (g) 729.67 ± 1.34 731.33 ± 1.67 730.33 ± 2.67 730.33 ± 1.75 0.53 Final weight (g) 1073.44 ± 13.79a 1050.02 ± 5.69a 1106.67 ± 13.78b 1142.86 ± 18.07c 0.01 Daily gain (g) 11.46 ± 0.22a 10.62 ± 0.18a 12.54 ± 0.17b 13.75 ± 0.18c 0.02 Feed conversion ratio 5.30a 5.62a 4.63b 4.11c 0.02 Parameters EP0 EP1 EP2 EP3 P-value Initial weight (g) 729.67 ± 1.34 731.33 ± 1.67 730.33 ± 2.67 730.33 ± 1.75 0.53 Final weight (g) 1073.44 ± 13.79a 1050.02 ± 5.69a 1106.67 ± 13.78b 1142.86 ± 18.07c 0.01 Daily gain (g) 11.46 ± 0.22a 10.62 ± 0.18a 12.54 ± 0.17b 13.75 ± 0.18c 0.02 Feed conversion ratio 5.30a 5.62a 4.63b 4.11c 0.02 Note: values are mean ± SE in each group. Values not sharing a common superscript differ significantly at P < 0.05. EP0, EP1, EP2, EP3 indicates diets supplemented with 0, 1%, 3%, or 5% earthworm powder, respectively. View Large Blood Characteristics Table 4 shows that the TP, ALB, GLB, BUN, SOD, ALKP, and CAT levels increased and the GOT, GPT, and CHE levels decreased during the experiment in both the control and the experimental group birds. The TP values in all the experimental groups differed from that in the control group (P = 0.03). The EP3 treatment group had higher (P < 0.05) GLB and SOD levels than the other treatment groups, whereas the GSH-Px and CAT levels were higher (P < 0.05) in the EP3 treatment group than those in the control group. In addition, nonsignificant differences in the GSH-Px and CAT levels were found between the control group and the EP1 and EP2 treatment groups. Table 4. Blood characteristics of broiler pullets fed earthworm powder. Parameters EP0 EP1 EP2 EP3 P-value TP1 (g/L) 56.26 ± 8.38a 66.59 ± 7.63b 62.81 ± 7.61b 66.79 ± 7.36b 0.03 ALB2 (g/L) 13.12 ± 1.09 14.42 ± 1.60 15.09 ± 2.46 13.01 ± 2.24 0.09 GLB3 (g/L) 43.14 ± 5.51a 52.17 ± 8.20a 47.72 ±6. 53a 53.78 ± 6.33b 0.04 GOT4 (U/L) 203.86 ± 8.15 213.35 ± 8.15 215.21 ± 9.78 184.87 ± 9.45 0.07 ALT5 (mg/L) 23.97 ± 9.50 27.21 ± 4.28 24.03 ± 6.37 22.99 ± 1.74 0.11 SOD6 (U/mL) 174.96 ± 7.37a 183.09 ± 6.51a 162.96 ± 6.86a 255.42 ± 6.86b 0.03 CHE7 (U/100 L) 81.97 ± 7.86 88.14 ± 6.01 80.02 ± 4.82 75.45 ± 3.71 0.08 ALKP8 (U/L) 75.21 ± 6.71 62.86 ± 8.96 78.78 ± 8.10 81.16 ± 9.12 0.06 GSH-PX9(umol/L) 454.68 ± 9.12a 484.65 ± 10.34a 494.18 ± 10.10a 524.55 ± 12.75b 0.03 CAT10 (U/L) 0.45 ± 0.01a 0.48 ± 0.01a 0.50 ± 0.01a 0.52 ± 0.02b 0.02 Parameters EP0 EP1 EP2 EP3 P-value TP1 (g/L) 56.26 ± 8.38a 66.59 ± 7.63b 62.81 ± 7.61b 66.79 ± 7.36b 0.03 ALB2 (g/L) 13.12 ± 1.09 14.42 ± 1.60 15.09 ± 2.46 13.01 ± 2.24 0.09 GLB3 (g/L) 43.14 ± 5.51a 52.17 ± 8.20a 47.72 ±6. 53a 53.78 ± 6.33b 0.04 GOT4 (U/L) 203.86 ± 8.15 213.35 ± 8.15 215.21 ± 9.78 184.87 ± 9.45 0.07 ALT5 (mg/L) 23.97 ± 9.50 27.21 ± 4.28 24.03 ± 6.37 22.99 ± 1.74 0.11 SOD6 (U/mL) 174.96 ± 7.37a 183.09 ± 6.51a 162.96 ± 6.86a 255.42 ± 6.86b 0.03 CHE7 (U/100 L) 81.97 ± 7.86 88.14 ± 6.01 80.02 ± 4.82 75.45 ± 3.71 0.08 ALKP8 (U/L) 75.21 ± 6.71 62.86 ± 8.96 78.78 ± 8.10 81.16 ± 9.12 0.06 GSH-PX9(umol/L) 454.68 ± 9.12a 484.65 ± 10.34a 494.18 ± 10.10a 524.55 ± 12.75b 0.03 CAT10 (U/L) 0.45 ± 0.01a 0.48 ± 0.01a 0.50 ± 0.01a 0.52 ± 0.02b 0.02 Note: values are mean ± SE in each group. Values not sharing a common superscript differ significantly at P < 0.05. EP0, EP 1, EP 2, EP3 indicates diets supplemented with 0, 1%, 3%, or 5% earthworm powder, respectively. 1Total protein. 2Albumin. 3Gamma-globulin. 4Glutamic oxaloacetic transaminase. 5Alanine aminotransferase. 6Superoxide dismutase. 7Cholinesterase. 8Alkaline phosphatase. 9Glutathione peroxidase. 10Catalase. View Large Table 4. Blood characteristics of broiler pullets fed earthworm powder. Parameters EP0 EP1 EP2 EP3 P-value TP1 (g/L) 56.26 ± 8.38a 66.59 ± 7.63b 62.81 ± 7.61b 66.79 ± 7.36b 0.03 ALB2 (g/L) 13.12 ± 1.09 14.42 ± 1.60 15.09 ± 2.46 13.01 ± 2.24 0.09 GLB3 (g/L) 43.14 ± 5.51a 52.17 ± 8.20a 47.72 ±6. 53a 53.78 ± 6.33b 0.04 GOT4 (U/L) 203.86 ± 8.15 213.35 ± 8.15 215.21 ± 9.78 184.87 ± 9.45 0.07 ALT5 (mg/L) 23.97 ± 9.50 27.21 ± 4.28 24.03 ± 6.37 22.99 ± 1.74 0.11 SOD6 (U/mL) 174.96 ± 7.37a 183.09 ± 6.51a 162.96 ± 6.86a 255.42 ± 6.86b 0.03 CHE7 (U/100 L) 81.97 ± 7.86 88.14 ± 6.01 80.02 ± 4.82 75.45 ± 3.71 0.08 ALKP8 (U/L) 75.21 ± 6.71 62.86 ± 8.96 78.78 ± 8.10 81.16 ± 9.12 0.06 GSH-PX9(umol/L) 454.68 ± 9.12a 484.65 ± 10.34a 494.18 ± 10.10a 524.55 ± 12.75b 0.03 CAT10 (U/L) 0.45 ± 0.01a 0.48 ± 0.01a 0.50 ± 0.01a 0.52 ± 0.02b 0.02 Parameters EP0 EP1 EP2 EP3 P-value TP1 (g/L) 56.26 ± 8.38a 66.59 ± 7.63b 62.81 ± 7.61b 66.79 ± 7.36b 0.03 ALB2 (g/L) 13.12 ± 1.09 14.42 ± 1.60 15.09 ± 2.46 13.01 ± 2.24 0.09 GLB3 (g/L) 43.14 ± 5.51a 52.17 ± 8.20a 47.72 ±6. 53a 53.78 ± 6.33b 0.04 GOT4 (U/L) 203.86 ± 8.15 213.35 ± 8.15 215.21 ± 9.78 184.87 ± 9.45 0.07 ALT5 (mg/L) 23.97 ± 9.50 27.21 ± 4.28 24.03 ± 6.37 22.99 ± 1.74 0.11 SOD6 (U/mL) 174.96 ± 7.37a 183.09 ± 6.51a 162.96 ± 6.86a 255.42 ± 6.86b 0.03 CHE7 (U/100 L) 81.97 ± 7.86 88.14 ± 6.01 80.02 ± 4.82 75.45 ± 3.71 0.08 ALKP8 (U/L) 75.21 ± 6.71 62.86 ± 8.96 78.78 ± 8.10 81.16 ± 9.12 0.06 GSH-PX9(umol/L) 454.68 ± 9.12a 484.65 ± 10.34a 494.18 ± 10.10a 524.55 ± 12.75b 0.03 CAT10 (U/L) 0.45 ± 0.01a 0.48 ± 0.01a 0.50 ± 0.01a 0.52 ± 0.02b 0.02 Note: values are mean ± SE in each group. Values not sharing a common superscript differ significantly at P < 0.05. EP0, EP 1, EP 2, EP3 indicates diets supplemented with 0, 1%, 3%, or 5% earthworm powder, respectively. 1Total protein. 2Albumin. 3Gamma-globulin. 4Glutamic oxaloacetic transaminase. 5Alanine aminotransferase. 6Superoxide dismutase. 7Cholinesterase. 8Alkaline phosphatase. 9Glutathione peroxidase. 10Catalase. View Large Antioxidant enzymes (SOD, CAT, and GSH-Px) protect the body from oxidative damage [25]. Superoxide dismutase and CAT can eliminate free radicals during xenobiotic exposure [29]. Glutathione peroxidase is a peroxide decomposition enzyme, which uses glutathione as a substrate to transform H2O2 into H2O [30]. Glutathione is a major nonprotein thiol in living organisms and plays a central role in coordinating the body's antioxidant defense mechanism. Prakash et al. [15] reported that earthworm powder exhibits potent antioxidant and hepatoprotective properties in alcohol-hepatotoxic rats. The present study showed that a diet containing 5% earthworm powder increased GLB, SOD, GSH-Px, and CAT activities in the pullets. These findings indicate that dietary earthworm powder supplementation can increase the antioxidant capacity of the body, and supplementing broiler diets with earthworm powder might improve the bird's antioxidant system. Nonsignificant differences were observed in the levels of ALB, GOT, GPT, and ALKP in all the treatment groups. The differences between the days 1 and 30 were not statistically significant with respect to any of the parameters we measured. Heavy Metal Residues The levels of the heavy metal residues in the chicken thigh muscles, chest muscles, and livers are shown in Table 5. The values measured in this study were compared with China's permissible limits given in the “General Standard of Contaminants in Foods” (GB 2762–2012). All the samples collected from the livers showed higher Cu, Zn, Pb, Cr, Cd, and As levels than the samples collected from both the groups of muscles. Table 5. Heavy metal residues of broiler pullets fed earthworm powder. Groups Item Species EP0 EP1 EP2 EP3 P-value Cu (mg/kg) Chest muscle 2.60 ± 0.39 2.40 ± 0.24 2.30 ± 0.72 2.51 ± 0.48 0.17 Thigh muscle 2.39 ± 0.41 2.47 ± 0.28 2.31 ± 0.37 2.45 ± 0.26 0.45 Liver 14.13 ± 1.63b 8.82 ± 1.57a 8.98 ± 1.67a 8.52 ± 1.83a 0.04 Zn (mg/kg) Chest muscle 16.79 ± 2.24 17.07 ± 3.11 15.30 ± 2.04 21.16 ± 3.87 0.06 Thigh muscle 57.91 ± 5.21 61.75 ± 3.62 65.97 ± 4.02 65.08 ± 3.97 0.06 Liver 94.63 ± 3.25 96.87 ± 2.81 97.84 ± 5.58 103.91 ± 6.73 0.09 Pb (mg/kg) Chest muscle 0.09 ± 0.02 0.09 ± 0.03 0.08 ± 0.03 0.09 ± 0.01 0.71 Thigh muscle ND ND ND ND Liver 0.42 ± 0.22 0.38 ± 0.13 0.45 ± 0.12 0.48 ± 0.22 0.23 Cr (μg/kg) Chest muscle 37.34 ± 6.03 44.56 ± 1.32 52.51 ± 2.55 49.60 ± 1.99 0.08 Thigh muscle 41.24 ± 3.61 56.84 ± 3.27 61.92 ± 4.52 45.57 ± 1.92 0.06 Liver 60.17 ± 2.16 68.61 ± 5.17 62.57 ± 1.44 58.74 ± 2.78 0.15 Cd (μg/kg) Chest muscle 16.65 ± 3.68 16.87 ± 4.14 12.58 ± 2.42 16.81 ± 1.62 0.21 Thigh muscle 12.80 ± 7.32 19.24 ± 4.61 ND ND 0.07 Liver 111.10 ± 8.03 142.40 ± 9.67 125.41 ± 4.23 183.53 ± 4.44 0.38 As (mg/kg) Chest muscle 0.21 ± 0.06 0.18 ± 0.03 0.22 ± 0.04 0.22 ± 0.03 0.36 Thigh muscle 0.28 ± 0.06a 0.24 ± 0.02a 0.34 ± 0.08b 0.26 ± 0.08a 0.02 Liver 2.52 ± 0.41 2.77 ± 0.47 4.76 ± 0.45 4.11 ± 1.18 0.07 Groups Item Species EP0 EP1 EP2 EP3 P-value Cu (mg/kg) Chest muscle 2.60 ± 0.39 2.40 ± 0.24 2.30 ± 0.72 2.51 ± 0.48 0.17 Thigh muscle 2.39 ± 0.41 2.47 ± 0.28 2.31 ± 0.37 2.45 ± 0.26 0.45 Liver 14.13 ± 1.63b 8.82 ± 1.57a 8.98 ± 1.67a 8.52 ± 1.83a 0.04 Zn (mg/kg) Chest muscle 16.79 ± 2.24 17.07 ± 3.11 15.30 ± 2.04 21.16 ± 3.87 0.06 Thigh muscle 57.91 ± 5.21 61.75 ± 3.62 65.97 ± 4.02 65.08 ± 3.97 0.06 Liver 94.63 ± 3.25 96.87 ± 2.81 97.84 ± 5.58 103.91 ± 6.73 0.09 Pb (mg/kg) Chest muscle 0.09 ± 0.02 0.09 ± 0.03 0.08 ± 0.03 0.09 ± 0.01 0.71 Thigh muscle ND ND ND ND Liver 0.42 ± 0.22 0.38 ± 0.13 0.45 ± 0.12 0.48 ± 0.22 0.23 Cr (μg/kg) Chest muscle 37.34 ± 6.03 44.56 ± 1.32 52.51 ± 2.55 49.60 ± 1.99 0.08 Thigh muscle 41.24 ± 3.61 56.84 ± 3.27 61.92 ± 4.52 45.57 ± 1.92 0.06 Liver 60.17 ± 2.16 68.61 ± 5.17 62.57 ± 1.44 58.74 ± 2.78 0.15 Cd (μg/kg) Chest muscle 16.65 ± 3.68 16.87 ± 4.14 12.58 ± 2.42 16.81 ± 1.62 0.21 Thigh muscle 12.80 ± 7.32 19.24 ± 4.61 ND ND 0.07 Liver 111.10 ± 8.03 142.40 ± 9.67 125.41 ± 4.23 183.53 ± 4.44 0.38 As (mg/kg) Chest muscle 0.21 ± 0.06 0.18 ± 0.03 0.22 ± 0.04 0.22 ± 0.03 0.36 Thigh muscle 0.28 ± 0.06a 0.24 ± 0.02a 0.34 ± 0.08b 0.26 ± 0.08a 0.02 Liver 2.52 ± 0.41 2.77 ± 0.47 4.76 ± 0.45 4.11 ± 1.18 0.07 Note: values are mean ± SE in each group. Values not sharing a common superscript differ significantly at P < 0.05. “ND” means no detection. EP0, EP 1, EP 2, and E3 indicates diets supplemented with 0, 1%, 3%, or 5% earthworm powder, respectively. View Large Table 5. Heavy metal residues of broiler pullets fed earthworm powder. Groups Item Species EP0 EP1 EP2 EP3 P-value Cu (mg/kg) Chest muscle 2.60 ± 0.39 2.40 ± 0.24 2.30 ± 0.72 2.51 ± 0.48 0.17 Thigh muscle 2.39 ± 0.41 2.47 ± 0.28 2.31 ± 0.37 2.45 ± 0.26 0.45 Liver 14.13 ± 1.63b 8.82 ± 1.57a 8.98 ± 1.67a 8.52 ± 1.83a 0.04 Zn (mg/kg) Chest muscle 16.79 ± 2.24 17.07 ± 3.11 15.30 ± 2.04 21.16 ± 3.87 0.06 Thigh muscle 57.91 ± 5.21 61.75 ± 3.62 65.97 ± 4.02 65.08 ± 3.97 0.06 Liver 94.63 ± 3.25 96.87 ± 2.81 97.84 ± 5.58 103.91 ± 6.73 0.09 Pb (mg/kg) Chest muscle 0.09 ± 0.02 0.09 ± 0.03 0.08 ± 0.03 0.09 ± 0.01 0.71 Thigh muscle ND ND ND ND Liver 0.42 ± 0.22 0.38 ± 0.13 0.45 ± 0.12 0.48 ± 0.22 0.23 Cr (μg/kg) Chest muscle 37.34 ± 6.03 44.56 ± 1.32 52.51 ± 2.55 49.60 ± 1.99 0.08 Thigh muscle 41.24 ± 3.61 56.84 ± 3.27 61.92 ± 4.52 45.57 ± 1.92 0.06 Liver 60.17 ± 2.16 68.61 ± 5.17 62.57 ± 1.44 58.74 ± 2.78 0.15 Cd (μg/kg) Chest muscle 16.65 ± 3.68 16.87 ± 4.14 12.58 ± 2.42 16.81 ± 1.62 0.21 Thigh muscle 12.80 ± 7.32 19.24 ± 4.61 ND ND 0.07 Liver 111.10 ± 8.03 142.40 ± 9.67 125.41 ± 4.23 183.53 ± 4.44 0.38 As (mg/kg) Chest muscle 0.21 ± 0.06 0.18 ± 0.03 0.22 ± 0.04 0.22 ± 0.03 0.36 Thigh muscle 0.28 ± 0.06a 0.24 ± 0.02a 0.34 ± 0.08b 0.26 ± 0.08a 0.02 Liver 2.52 ± 0.41 2.77 ± 0.47 4.76 ± 0.45 4.11 ± 1.18 0.07 Groups Item Species EP0 EP1 EP2 EP3 P-value Cu (mg/kg) Chest muscle 2.60 ± 0.39 2.40 ± 0.24 2.30 ± 0.72 2.51 ± 0.48 0.17 Thigh muscle 2.39 ± 0.41 2.47 ± 0.28 2.31 ± 0.37 2.45 ± 0.26 0.45 Liver 14.13 ± 1.63b 8.82 ± 1.57a 8.98 ± 1.67a 8.52 ± 1.83a 0.04 Zn (mg/kg) Chest muscle 16.79 ± 2.24 17.07 ± 3.11 15.30 ± 2.04 21.16 ± 3.87 0.06 Thigh muscle 57.91 ± 5.21 61.75 ± 3.62 65.97 ± 4.02 65.08 ± 3.97 0.06 Liver 94.63 ± 3.25 96.87 ± 2.81 97.84 ± 5.58 103.91 ± 6.73 0.09 Pb (mg/kg) Chest muscle 0.09 ± 0.02 0.09 ± 0.03 0.08 ± 0.03 0.09 ± 0.01 0.71 Thigh muscle ND ND ND ND Liver 0.42 ± 0.22 0.38 ± 0.13 0.45 ± 0.12 0.48 ± 0.22 0.23 Cr (μg/kg) Chest muscle 37.34 ± 6.03 44.56 ± 1.32 52.51 ± 2.55 49.60 ± 1.99 0.08 Thigh muscle 41.24 ± 3.61 56.84 ± 3.27 61.92 ± 4.52 45.57 ± 1.92 0.06 Liver 60.17 ± 2.16 68.61 ± 5.17 62.57 ± 1.44 58.74 ± 2.78 0.15 Cd (μg/kg) Chest muscle 16.65 ± 3.68 16.87 ± 4.14 12.58 ± 2.42 16.81 ± 1.62 0.21 Thigh muscle 12.80 ± 7.32 19.24 ± 4.61 ND ND 0.07 Liver 111.10 ± 8.03 142.40 ± 9.67 125.41 ± 4.23 183.53 ± 4.44 0.38 As (mg/kg) Chest muscle 0.21 ± 0.06 0.18 ± 0.03 0.22 ± 0.04 0.22 ± 0.03 0.36 Thigh muscle 0.28 ± 0.06a 0.24 ± 0.02a 0.34 ± 0.08b 0.26 ± 0.08a 0.02 Liver 2.52 ± 0.41 2.77 ± 0.47 4.76 ± 0.45 4.11 ± 1.18 0.07 Note: values are mean ± SE in each group. Values not sharing a common superscript differ significantly at P < 0.05. “ND” means no detection. EP0, EP 1, EP 2, and E3 indicates diets supplemented with 0, 1%, 3%, or 5% earthworm powder, respectively. View Large There was no significant difference in the Cu levels between the samples collected from the thigh and chest muscles of groups EP1, EP2, and EP3. The concentrations of Cu in the liver samples exhibited significant differences (P = 0.04) between EP0 and the other groups. This difference might have been caused by the increase in the SOD level with the addition of earthworm powder. Because Cu is an important component of SOD [31], as the SOD level increased, the Cu level in the liver decreased correspondingly to form Cu-SOD. The Cu levels in all the samples were below China's permissible limit. The mean Zn levels in each tissue type showed no significant differences among the treatment groups (P > 0.05). The highest Zn levels of 94.63 ± 3.25, 96.87 ± 2.81, 97.84 ± 5.58, and 103.91 ± 6.73 mg kg−1 were all detected in the liver samples, which indicated that the liver is possibly the main Zn accumulation area. All the values in the studied samples were below China's permissible limit. There were nonsignificant differences in the levels of Pb, Cr, and Cd among all the treatment groups, with the concentrations of all the 3 heavy metals generally <0.5 mg kg−1 in the samples from all the groups. The levels found in this study were much lower than China's permissible limits of 1 mg/kg. However, there were relatively high levels of As (2.52 ± 0.41, 2.77 ± 0.47, 4.76 ± 0.45, and 4.11 ± 1.18 mg/kg in EP0, EP1, EP2, and EP3, respectively) in the liver. There is no permissible limit for As listed in the National Food Safety Standard GB 2762–2012 of the People's Republic of China. In general, the liver contained higher levels of heavy metals than did the thigh and chest muscles. All the heavy metal residues in the thigh muscle, chest muscle, and liver were below China's permissible limits. CONCLUSIONS AND APPLICATIONS Dietary earthworm powder supplementation can increase the antioxidant capacity of the body, and supplementation of broiler diets with earthworm powder might improve the antioxidant system of the broilers. Heavy metals in poultry diets are maintained below the permitted levels based on the National Feed Hygiene Standard GB 13078–2001 of the People's Republic of China. The residues of all the heavy metals in the thigh, chest muscles, and liver of the broiler pullets, following supplementation of the broiler diet with earthworm powder, were below China's permissible limits. Therefore, earthworm powder can be used as a feed supplement in broiler diets. Note Primary Audience: Nutritionists, Quality Assurance Personnel, Researchers, Veterinarians REFERENCES AND NOTES 1. Ravindran V. , Blair R. . 1993 . Feed resources for poultry production in Asia and the Pacific. III. Animal protein sources . Worlds Poult. Sci. J. 49 : 219 – 235 . 2. Sogbesan A. O. , Ugwumba A. A. A. . 2008 . Nutritional evaluation of termite (Macrotermes subhyalinus) meal as animal protein supplements in the diets of Heterobranchus longifilis (Valenciennes, 1840) fingerlings . Turk. J. Fish. Aquat. Sci. 8 : 149 – 157 . 3. Bahadori Z. , Esmaielzadeh L. , Karimi-Torshizi M. A. , Seidavi A. , Olivares J. , Rojas S. , Salem A.Z.M. , Khusrof A. , López S. . 2017 . The effect of earthworm (Eisenia foetida) meal with vermi-humus on growth performance, hematology, immunity, intestinal microbiota, carcass characteristics, and meat quality of broiler chickens . Livestock Science 202 : 74 – 81 . 4. Sogbesan O. A. , Ugwumba A. A. A. . 2006 . Effect of different substrates on growth and productivity of Nigeria semi-arid zone earthworm (Hyperiodrilus euryaulos, Clausen, 1842)(Oligochaeta: Eudrilinae) . World J. Zool. 103 – 112 . 5. Istiqomah L. , Sofyan A. , Damayanti E. , Julendra H. . 2009 . Amino acid profile of earthworm and earthworm meal (Lumbricus rubellus) for animal feedstuff . J. Indonesian Trop. Anim. Agric. 34 : 253 – 257 . 6. Bahadori Z. , Esmaylzadeh L. , Torshizi M. A. K. . 2015 . The effect of earthworm (Eisenia foetida) and vermihumus meal in diet on broilers chicken efficiency and carcass components . Biol. Form. 7 : 998 – 1005 . 7. Jang Ho S . 2009 . The study on treatment of poultry manure by eartworm and earthworm meal on the performance of broilers and laying hens and safety of meat and egg . Korean J. Org. Agric. 17 : 63 – 82 . 8. Atiyeh R. M. , Arancon N. , Edwards C. A. , Metzger J. D. . 2000 . Influence of earthworm-processed pig manure on the growth and yield of greenhouse tomatoes . Bioresour. Technol. 75 : 175 – 180 . 9. Hansen M. , Björklund E. , Popovic O. , Jensen L. S. , Jacobsen C. S. , Sedlak D. L. , Halling-Sørensen B. . 2015 . Animal manure separation technologies diminish the environmental burden of steroid hormones . Environ. Sci. Technol. Lett. 2 : 133 – 137 . 10. Helmke P. A. , Robarge W. P. , Korotev R. L. , Schomberg P. J. . 1979 . Effects of soil-applied sewage sludge on concentrations of elements in earthworms . J. Environ. Qual. 8 : 322 – 327 . 11. Demirezen D. , Aksoy A. . 2006 . Heavy metal levels in vegetables in Turkey are within safe limits for Cu, Zn, Ni and exceeded for Cd and Pb . J. Food Qual. 29 : 252 – 265 . 12. Bokori J. , Fekete S. , Glavits R. , Kadar I. , Koncz J. , Kövári L. 1995 . Complex study of the physiological role of cadmium. IV. Effects of prolonged dietary exposure of broiler chickens to cadmium . Acta. Vet. Hung. 44 : 57 – 74 . 13. Mariam I. , Iqbal S. , Nagra S. A. , 2004 . Distribution of some trace and macrominerals in beef, mutton and poultry . Int. J. Agric. Biol. 6 : 816 – 820 . 14. Ibáñez I. A. , Herrera C. A. , Velásquez L. A. , Hebel P. . 1993 . Nutritional and toxicological evaluation on rats of earthworm (Eisenia fetida) meal as protein source for animal feed . Anim. Feed Sci. Technol. 42 : 165 – 172 . 15. Prakash M. , Balamurugan M. , Parthasarathi K. , Gunasekaran G. , Cooper E. L. , Ranganathan L. . 2007 . Anti-ulceral and anti-oxidative properties of “earthworm paste” of Lampito mauritii (Kinberg) . Eur. Rev. Med. Pharmacol. Sci. 11 : 9 – 15 . 16. Mittler R. 2002 . Oxidative stress, antioxidants and stress tolerance . Trends Plant Sci. 7 : 405 – 410 . 17. Xinfeng Biological Science and Technology Co., Ltd. Nanchang, CHN . 18. Prakash M. , Gunasekaran G. , Elumalai K. . 2008 . Effect of earthworm powder on antioxidant enzymes in alcohol induced hepatotoxic rats . Eur. Rev. Med. Pharmacol. Sci. 12 : 237 – 243 . 19. Quemener B. , Thibault J. F. . 1990 . Assessment of methanolysis for the determination of sugars in pectins . Carbohydr. Res. 206 : 277 – 287 . 20. Amino acid auto analyzer L-8900, Hitachi Ltd., JPN . 21. Spectrophotometry, ICP-OES 5300DV, PerkinElmer Company, Waltham, MA . 22. Commercial laboratory kits, Nanjing Jiancheng Bioengineering Institute, CHN . 23. Spectrophotometer, Multiskan GO, Thermo Scientific, FI . 24. IBM SPSS 19.0, IBM, Armonk, NY . 25. Tuan N. N. , Focken U. . 2009 . Earthworm powder as potential protein source in diets for common carp (Cyprinus carpio L.). Biophysical and socio-economic frame conditions for the sustainable management of natural resources. Hamburg: Tropentag . 26. Prayogi H. S. 2011 . The effect of earthworm meal supplementation in the diet on quail's growth performance in attempt to replace the usage of fish meal . Int. J. of Poul. Sci. 10 : 804 – 806 . 27. Taboga L. 1980 . The nutritional value of earthworms for chickens . Br. Poult. Sci. 21 : 405 – 410 . 28. Julendra H. , Damayanti E. , Lusty Istiqomah S. N. , Karimy M. F. . 2012 . The effectiveness of earthworm meal supplementation as antibiotic growth promoter replacer with different processing method . The 1st Poult. Int. Sem. 146 – 157 . 29. Ognjanovic B. I. , Markovic S. D. , Pavlovic S. Z. , Zikic R. V. , Stajn A. S. , Saicic Z. S. . 2008 . Effect of chronic cadmium exposure on antioxidant defense system in some tissues of rats: protective effect of selenium . Physiol. Res. 57 : 403 . 30. Tezcan Ö. , Pandır D. , Baş H. . 2012 . The effects of cadmium on enzymatic antioxidant system and lipid peroxidation of human erythrocytes in vitro and the protective role of plasma level of vitamins C and E . Pol. J. Environ. Stud. 21 . 31. Hafeman D. G. , Sunde R. A. , Hoekstra W. G. . 1974 . Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat . J. Nutr. 104 : 580 – 587 . 32. Baret A. , Jadot G. , Michelson A. M. . 1984 . Pharmacokinetic and anti-inflammatory properties in the rat of superoxide dismutases (Cu SODs and Mn SOD) from various species . Biochem. Pharmacol. 33 : 2755 – 2760 . Acknowledgements This work was supported by the Fund for National science and technology support plan (2012BAD14B14), Science and Technology Plan Projects of Department of Education of Jiangxi Province (grant number: GJJ160401). © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Poultry Research Oxford University Press

Effects of Dietary Supplementation With Earthworm Powder on Production Performance, Blood Characteristics, and Heavy Metal Residues of Broiler Pullets

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© 2018 Poultry Science Association Inc.
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Abstract

SUMMARY Earthworms have been studied as a feed additive and animal protein source for poultry for many years. The interest in earthworm use for manure management has greatly increased over the past few decades. However, it is unknown whether earthworm powder in the diet poses a potential heavy metal risk for broiler pullets. This study was conducted to investigate the safety of adding earthworm powder in the diets of broiler pullets. A total of 240 Ningdu Yellow broiler pullets were randomly assigned to 4 groups receiving diets supplemented with 0%, 1%, 3%, or 5% earthworm powder for 30 d. Feed conversion ratios were improved by 12.64% and 22.45%, when 3% and 5% earthworm powder, respectively, were added (P = 0.02). Supplementing the diet with 5% earthworm powder had no negative effects on the growth of pullets, and increased antioxidant enzyme (superoxide dismutase, catalase, and glutathione peroxidase) activities in the liver (P < 0.05). The liver contained higher levels of heavy metals than did the thigh and chest muscles. All heavy metal residues in the thigh and chest muscles, as well as the liver, were below China's permissible limits. These results suggest that dietary supplementation with 1%–5% earthworm powder is safe for broiler pullets. DESCRIPTION OF PROBLEM The presence of animal protein in poultry feed is essential for the growth and health of the reared birds. Fish meal, because of its balanced amino acid contents, is the conventional source of animal protein in poultry feeds [1]. However, because of the increasing cost of high-quality fish meals and due to the declining stocks of fish obtained from capture fishery, at present, there is a need to search for alternative sources of animal protein for incorporation into poultry feeds [2]. Earthworm meal is an easily available resource that can be used as a feed additive and an animal protein source for poultry [3, 4]. Many scientists have reported that the rapid breeding and composting potential of earthworms may be effectively utilized as an economic animal feed protein for the fish and poultry industries worldwide [3, 4, 5]. Bahadori et al. [6] showed that earthworm meal is rich in lysine (4.44%), methionine (1.20%), and metabolizable energy (3,258 KcalME/kg). Jang Ho [7] observed that using earthworm meal to feed broiler chickens could improve feed intake, protein digestibility, and weight gain. Over the last few decades, as regulations for the disposal of animal manure have become more rigorous [8, 9], interest in the use of earthworms as an ecologically sound method of manure management has increased dramatically. Earthworms, many cultured on livestock manure, can take up and accumulate heavy metals, such as cadmium, mercury, and gold, in their tissues [10]. As such, even if the levels of heavy metals in poultry diets are maintained below the permitted levels based on the National Feed Hygiene Standard GB 13078–2001 of the People's Republic of China, addition of heavy metal-contaminated earthworm meals in the poultry diets may lead to the deposition of heavy metals in the poultry carcasses; these metals can then be transferred to humans via consumption of the contaminated meat. Contamination with heavy metals is a serious toxicity threat, since they are capable of bioaccumulation and biomagnification in the food chain [11]. These pollutants have direct toxic effects on the physiology because they are stored or incorporated into tissues, sometimes permanently [12, 13]. Ibáñez et al. [14] evaluated the toxicology of earthworm (Eisenia fetida) meals used as a protein source in rats. The continued development of intensive livestock production systems has led to an increased heavy metal contamination of livestock manures, thereby increasing the importance of evaluating the heavy metal contents in the broiler chickens fed earthworm meal. Earthworms have been widely used in traditional medicine for thousands of years, as they possess rich natural sources of antioxidants [15]. Supplementation of diets with earthworm meal powder (henceforth earthworm powder) might improve the antioxidant system of broilers, which may protect them from the oxidative damage inflicted by excess reactive oxygen species that otherwise promote cell injury and death in a broad variety of disorders, and are considered to cause important pathophysiological conditions [16]. Thus, it is necessary to investigate the possible effects of earthworm powder used as a feed additive on some antiperoxidative enzymes present in the blood serum of the broilers. The following experiments were conducted to evaluate the heavy metal concentrations and antioxidants in the blood serum resulting from the addition of earthworm powder to the diets of broiler pullets. Specifically, our objectives were to evaluate the effects of earthworm powder on production performance, blood characteristics, and heavy metal contents of the broiler pullet carcass. MATERIALS AND METHODS Preparation of Earthworm Powder Earthworm powder was prepared following the method of Ibáñez et al. [14]. The cultivated earthworms used in this experiment were grown in a pig manure–wheat straw mixture [17]. Moisture, crude protein, and metabolic energy of the powder were determined by the usual feed procedures [18], and crude fat by the Association of Official Analytical Chemists method [19]. Amino acid composition was determined with an amino acid auto analyzer [20]. The powder was analyzed for total heavy metals (Zn, Cu, Pb, Cd, As, and Cr) by aqua regia digestion and analysis using inductively coupled plasma spectrophotometry [21] or graphite furnace atomic absorption spectrophotometry (GFAAS). Experimental Design The care of laboratory animals and the experimental methods involving animals were approved by Jiangxi Agricultural University Institutional Animal Care and Use Committee. A total of 240, 9-wk-old Ningdu Yellow broiler pullets were randomly assigned to one of 4 treatments in an experiment that lasted 30 d. There were 5 replicates (12 pullets per replicate) for each treatment. Pullets were weighed individually at the onset of the experiment and then assigned to treatments in a randomized block design. Pullets were provided with free access to water through the nipple of an automatic drinker and feed through a common trough feeder. The composition of the corn and soybean meal basal diet is shown in Table 1, and the heavy metal contents in the diets were below China's permissible limits (GB13078–2001). Dietary treatments consisted of the corn and soybean meal basal diet supplemented with 0% (control EP 0), 1% (EP 1), 3% (EP 2), or 5% earthworm powder (EP3; Table 1). To balance the protein content of the diet, the proportion of soybean was decreased with the increasing earthworm powder content. Because earthworm meal is rich in lysine and digestible energy (DE), as evident from the estimated nutrient proportions of the earthworm powder (Table 2), the nutrient level of threonine and DE in the diet also increased in correspondence with the supplementation of earthworm powder (Table 1). The experimental diets were formulated in accordance with standard chicken breed recommendations (NY/T33-2004). Before the beginning of the experiment, 9-wk-old hens were purchased and provided with the basal diet for a 7-wk adjustment period. Feed intake and body weight were recorded at the end of the experiment (30 d). Table 1. Composition of diet. Groups Items EP0 EP1 EP2 EP3 Diet composition (%) Corn 68.20 68.20 68.20 68.20 Soybean bean 21.30 20.16 17.87 15.58 Wheat bran 4.00 4.14 4.43 4.72 Earthworm powder 0 1 3 5 Limestone 1.10 1.10 1.10 1.10 Dicalcium phosphate 1.40 1.40 1.40 1.40 Oili 2.70 2.70 2.70 2.70 Sault 0.30 0.30 0.30 0.30 1% Premix 1 1 1 1 Nutrient level DE (MJ/kg) 12.98 13.38 14.16 14.95 Crude protein (%) 16.00 16.00 16.00 16.00 Met + Cys (%) 0.69 0.69 0.69 0.70 Lys (%) 0.79 0.80 0.82 0.83 Thr (%) 0.70 0.71 0.72 0.73 Calcium (%) 0.82 0.82 0.81 0.81 Total P (%) 0.59 0.58 0.57 0.56 Available P (%) 0.30 0.29 0.29 0.29 Cu (mg/kg) 9.26 7.23 13.59 13.41 Zn (mg/kg) 79.08 104.39 102.47 105.46 Cr (mg/kg) 0.25 0.45 1.07 1.84 Cd (mg/kg) 0.03 0.04 0.08 0.07 As (mg/kg) 3.97 3.76 5.80 3.68 Pb(mg/kg) 1.17 1.31 1.67 1.77 Groups Items EP0 EP1 EP2 EP3 Diet composition (%) Corn 68.20 68.20 68.20 68.20 Soybean bean 21.30 20.16 17.87 15.58 Wheat bran 4.00 4.14 4.43 4.72 Earthworm powder 0 1 3 5 Limestone 1.10 1.10 1.10 1.10 Dicalcium phosphate 1.40 1.40 1.40 1.40 Oili 2.70 2.70 2.70 2.70 Sault 0.30 0.30 0.30 0.30 1% Premix 1 1 1 1 Nutrient level DE (MJ/kg) 12.98 13.38 14.16 14.95 Crude protein (%) 16.00 16.00 16.00 16.00 Met + Cys (%) 0.69 0.69 0.69 0.70 Lys (%) 0.79 0.80 0.82 0.83 Thr (%) 0.70 0.71 0.72 0.73 Calcium (%) 0.82 0.82 0.81 0.81 Total P (%) 0.59 0.58 0.57 0.56 Available P (%) 0.30 0.29 0.29 0.29 Cu (mg/kg) 9.26 7.23 13.59 13.41 Zn (mg/kg) 79.08 104.39 102.47 105.46 Cr (mg/kg) 0.25 0.45 1.07 1.84 Cd (mg/kg) 0.03 0.04 0.08 0.07 As (mg/kg) 3.97 3.76 5.80 3.68 Pb(mg/kg) 1.17 1.31 1.67 1.77 Note: The premix fodder contains: methionine, 1.0 g/kg; lysine, 0.7 g/kg; threonine, 0.8 g/kg; choline chloride, 0.5 g/kg; VA, 19,000 IU/kg; VD3, 5200 IU/kg; VE, 38 IU/kg; VB12, 0.022 mg/kg; VB6, 5.6 mg/kg; VK3, 3.8 mg/kg; riboflavin, 11.4 mg/kg; VB1, 8 mg/kg; D-pantothenic acid, 22.8 mg/kg; nicotinic acid, 38 mg/kg; biotin, 0.15 mg/kg; folic acid, 0.56 mg/kg; Mn, 85 mg/kg; Cu, 10 mg/kg; iodine, 0.35 mg/kg. View Large Table 1. Composition of diet. Groups Items EP0 EP1 EP2 EP3 Diet composition (%) Corn 68.20 68.20 68.20 68.20 Soybean bean 21.30 20.16 17.87 15.58 Wheat bran 4.00 4.14 4.43 4.72 Earthworm powder 0 1 3 5 Limestone 1.10 1.10 1.10 1.10 Dicalcium phosphate 1.40 1.40 1.40 1.40 Oili 2.70 2.70 2.70 2.70 Sault 0.30 0.30 0.30 0.30 1% Premix 1 1 1 1 Nutrient level DE (MJ/kg) 12.98 13.38 14.16 14.95 Crude protein (%) 16.00 16.00 16.00 16.00 Met + Cys (%) 0.69 0.69 0.69 0.70 Lys (%) 0.79 0.80 0.82 0.83 Thr (%) 0.70 0.71 0.72 0.73 Calcium (%) 0.82 0.82 0.81 0.81 Total P (%) 0.59 0.58 0.57 0.56 Available P (%) 0.30 0.29 0.29 0.29 Cu (mg/kg) 9.26 7.23 13.59 13.41 Zn (mg/kg) 79.08 104.39 102.47 105.46 Cr (mg/kg) 0.25 0.45 1.07 1.84 Cd (mg/kg) 0.03 0.04 0.08 0.07 As (mg/kg) 3.97 3.76 5.80 3.68 Pb(mg/kg) 1.17 1.31 1.67 1.77 Groups Items EP0 EP1 EP2 EP3 Diet composition (%) Corn 68.20 68.20 68.20 68.20 Soybean bean 21.30 20.16 17.87 15.58 Wheat bran 4.00 4.14 4.43 4.72 Earthworm powder 0 1 3 5 Limestone 1.10 1.10 1.10 1.10 Dicalcium phosphate 1.40 1.40 1.40 1.40 Oili 2.70 2.70 2.70 2.70 Sault 0.30 0.30 0.30 0.30 1% Premix 1 1 1 1 Nutrient level DE (MJ/kg) 12.98 13.38 14.16 14.95 Crude protein (%) 16.00 16.00 16.00 16.00 Met + Cys (%) 0.69 0.69 0.69 0.70 Lys (%) 0.79 0.80 0.82 0.83 Thr (%) 0.70 0.71 0.72 0.73 Calcium (%) 0.82 0.82 0.81 0.81 Total P (%) 0.59 0.58 0.57 0.56 Available P (%) 0.30 0.29 0.29 0.29 Cu (mg/kg) 9.26 7.23 13.59 13.41 Zn (mg/kg) 79.08 104.39 102.47 105.46 Cr (mg/kg) 0.25 0.45 1.07 1.84 Cd (mg/kg) 0.03 0.04 0.08 0.07 As (mg/kg) 3.97 3.76 5.80 3.68 Pb(mg/kg) 1.17 1.31 1.67 1.77 Note: The premix fodder contains: methionine, 1.0 g/kg; lysine, 0.7 g/kg; threonine, 0.8 g/kg; choline chloride, 0.5 g/kg; VA, 19,000 IU/kg; VD3, 5200 IU/kg; VE, 38 IU/kg; VB12, 0.022 mg/kg; VB6, 5.6 mg/kg; VK3, 3.8 mg/kg; riboflavin, 11.4 mg/kg; VB1, 8 mg/kg; D-pantothenic acid, 22.8 mg/kg; nicotinic acid, 38 mg/kg; biotin, 0.15 mg/kg; folic acid, 0.56 mg/kg; Mn, 85 mg/kg; Cu, 10 mg/kg; iodine, 0.35 mg/kg. View Large Table 2. Nutrient Ingredient of Earthworm Powder. Composition Earthworm powder Crude protein (%) 57.85 Crude fat (%) 3.50 Total amino acids (%) 50.80 Total essential amino acids (%) 38.31 Lys (%) 4.04 Thr (%) 2.64 DE (MJ/kg) 12.46 Composition Earthworm powder Crude protein (%) 57.85 Crude fat (%) 3.50 Total amino acids (%) 50.80 Total essential amino acids (%) 38.31 Lys (%) 4.04 Thr (%) 2.64 DE (MJ/kg) 12.46 View Large Table 2. Nutrient Ingredient of Earthworm Powder. Composition Earthworm powder Crude protein (%) 57.85 Crude fat (%) 3.50 Total amino acids (%) 50.80 Total essential amino acids (%) 38.31 Lys (%) 4.04 Thr (%) 2.64 DE (MJ/kg) 12.46 Composition Earthworm powder Crude protein (%) 57.85 Crude fat (%) 3.50 Total amino acids (%) 50.80 Total essential amino acids (%) 38.31 Lys (%) 4.04 Thr (%) 2.64 DE (MJ/kg) 12.46 View Large Sampling and Measurements A total of 10 birds were selected at random from each treatment on the initial day of the experiment. Blood samples were collected from the wing vein of each pullet on the initial and final days of the experiment, using a sterilized syringe and both nonheparinized tubes and K3EDTA vacuum tubes to obtain serum and whole blood, respectively. The blood samples were then centrifuged at 2000 × g at 4°C for 20 min within 1 h of collection to separate the serum. The total protein (TP), albumin, glutamic oxaloacetic transaminase (GOT), glutathione peroxidase (GSH-Px), alkaline phosphatase (ALKP), superoxide dismutase (SOD), catalase (CAT), alanine aminotransferase (ALT), and cholinesterase (CHE) were measured using commercial laboratory kits [22] with a spectrophotometer [23]. On the final day of the experiment, all the pullets were killed by decapitation, and samples (thigh muscles, chest muscles, and liver) were collected in polyethylene bags, stored at –18°C, and analyzed as soon as possible. The heavy metal contents in the homogenated tissues were estimated using inductively coupled plasma spectrophotometry or GFAAS. Statistical Analysis Analysis of variance was performed to analyze the data using IBM SPSS 19.0 [24]. Tukey's HSD was used in the analysis for multiple comparisons. The significance level was set at P < 0.05. RESULTS AND DISCUSSION Production Performance Table 3 summarizes the effects of dietary earthworm powder supplementation treatments on the enhancement of body weight, feed intake, and feed conversion ratio in 9-wk-old pullets. The measurements of all these parameters increased with the increasing proportion of the earthworm powder added. The feed conversion ratio was improved by 12.64% and 22.45% when 3% and 5% earthworm powder, respectively, were added (P = 0.02). Earthworm meal is equal to or surpasses other feed supplements, like fish meal and meat meal, as an important animal protein source for poultry. Table 1 showed that the nutrient level of threonine and DE in the diet increased in correspondence with the supplementation of earthworm powder. These results were in accordance with the findings of Bahadori et al. [6]. They showed that earthworm meal is rich in lysine and metabolizable energy, which may be the cause of improved feed conversion. Tuan [25] indicated that fish fed with diets containing earthworm powder had similar or higher growth rates, protein efficiency, and energy retention than that of a control group fishes. Prayogi [26] reported that weight gain and the feed-conversion ratio improved in quails fed diets supplemented with 10% earthworm meal. Taboga [27] indicated that the amino acid composition of earthworms was close to the amino acid requirements of chickens. In addition, Julendra et al. [28] indicted that the improvement in the body weight gain in broilers fed earthworm meal may have been caused by the antibacterial characteristics of the earthworm's coelomic fluid, which had the potential to thin the intestinal mucosa and increase nutrient absorption and feed efficiency [3]. Table 3. Growth Performance of broiler pullets fed earthworm powder. Parameters EP0 EP1 EP2 EP3 P-value Initial weight (g) 729.67 ± 1.34 731.33 ± 1.67 730.33 ± 2.67 730.33 ± 1.75 0.53 Final weight (g) 1073.44 ± 13.79a 1050.02 ± 5.69a 1106.67 ± 13.78b 1142.86 ± 18.07c 0.01 Daily gain (g) 11.46 ± 0.22a 10.62 ± 0.18a 12.54 ± 0.17b 13.75 ± 0.18c 0.02 Feed conversion ratio 5.30a 5.62a 4.63b 4.11c 0.02 Parameters EP0 EP1 EP2 EP3 P-value Initial weight (g) 729.67 ± 1.34 731.33 ± 1.67 730.33 ± 2.67 730.33 ± 1.75 0.53 Final weight (g) 1073.44 ± 13.79a 1050.02 ± 5.69a 1106.67 ± 13.78b 1142.86 ± 18.07c 0.01 Daily gain (g) 11.46 ± 0.22a 10.62 ± 0.18a 12.54 ± 0.17b 13.75 ± 0.18c 0.02 Feed conversion ratio 5.30a 5.62a 4.63b 4.11c 0.02 Note: values are mean ± SE in each group. Values not sharing a common superscript differ significantly at P < 0.05. EP0, EP1, EP2, EP3 indicates diets supplemented with 0, 1%, 3%, or 5% earthworm powder, respectively. View Large Table 3. Growth Performance of broiler pullets fed earthworm powder. Parameters EP0 EP1 EP2 EP3 P-value Initial weight (g) 729.67 ± 1.34 731.33 ± 1.67 730.33 ± 2.67 730.33 ± 1.75 0.53 Final weight (g) 1073.44 ± 13.79a 1050.02 ± 5.69a 1106.67 ± 13.78b 1142.86 ± 18.07c 0.01 Daily gain (g) 11.46 ± 0.22a 10.62 ± 0.18a 12.54 ± 0.17b 13.75 ± 0.18c 0.02 Feed conversion ratio 5.30a 5.62a 4.63b 4.11c 0.02 Parameters EP0 EP1 EP2 EP3 P-value Initial weight (g) 729.67 ± 1.34 731.33 ± 1.67 730.33 ± 2.67 730.33 ± 1.75 0.53 Final weight (g) 1073.44 ± 13.79a 1050.02 ± 5.69a 1106.67 ± 13.78b 1142.86 ± 18.07c 0.01 Daily gain (g) 11.46 ± 0.22a 10.62 ± 0.18a 12.54 ± 0.17b 13.75 ± 0.18c 0.02 Feed conversion ratio 5.30a 5.62a 4.63b 4.11c 0.02 Note: values are mean ± SE in each group. Values not sharing a common superscript differ significantly at P < 0.05. EP0, EP1, EP2, EP3 indicates diets supplemented with 0, 1%, 3%, or 5% earthworm powder, respectively. View Large Blood Characteristics Table 4 shows that the TP, ALB, GLB, BUN, SOD, ALKP, and CAT levels increased and the GOT, GPT, and CHE levels decreased during the experiment in both the control and the experimental group birds. The TP values in all the experimental groups differed from that in the control group (P = 0.03). The EP3 treatment group had higher (P < 0.05) GLB and SOD levels than the other treatment groups, whereas the GSH-Px and CAT levels were higher (P < 0.05) in the EP3 treatment group than those in the control group. In addition, nonsignificant differences in the GSH-Px and CAT levels were found between the control group and the EP1 and EP2 treatment groups. Table 4. Blood characteristics of broiler pullets fed earthworm powder. Parameters EP0 EP1 EP2 EP3 P-value TP1 (g/L) 56.26 ± 8.38a 66.59 ± 7.63b 62.81 ± 7.61b 66.79 ± 7.36b 0.03 ALB2 (g/L) 13.12 ± 1.09 14.42 ± 1.60 15.09 ± 2.46 13.01 ± 2.24 0.09 GLB3 (g/L) 43.14 ± 5.51a 52.17 ± 8.20a 47.72 ±6. 53a 53.78 ± 6.33b 0.04 GOT4 (U/L) 203.86 ± 8.15 213.35 ± 8.15 215.21 ± 9.78 184.87 ± 9.45 0.07 ALT5 (mg/L) 23.97 ± 9.50 27.21 ± 4.28 24.03 ± 6.37 22.99 ± 1.74 0.11 SOD6 (U/mL) 174.96 ± 7.37a 183.09 ± 6.51a 162.96 ± 6.86a 255.42 ± 6.86b 0.03 CHE7 (U/100 L) 81.97 ± 7.86 88.14 ± 6.01 80.02 ± 4.82 75.45 ± 3.71 0.08 ALKP8 (U/L) 75.21 ± 6.71 62.86 ± 8.96 78.78 ± 8.10 81.16 ± 9.12 0.06 GSH-PX9(umol/L) 454.68 ± 9.12a 484.65 ± 10.34a 494.18 ± 10.10a 524.55 ± 12.75b 0.03 CAT10 (U/L) 0.45 ± 0.01a 0.48 ± 0.01a 0.50 ± 0.01a 0.52 ± 0.02b 0.02 Parameters EP0 EP1 EP2 EP3 P-value TP1 (g/L) 56.26 ± 8.38a 66.59 ± 7.63b 62.81 ± 7.61b 66.79 ± 7.36b 0.03 ALB2 (g/L) 13.12 ± 1.09 14.42 ± 1.60 15.09 ± 2.46 13.01 ± 2.24 0.09 GLB3 (g/L) 43.14 ± 5.51a 52.17 ± 8.20a 47.72 ±6. 53a 53.78 ± 6.33b 0.04 GOT4 (U/L) 203.86 ± 8.15 213.35 ± 8.15 215.21 ± 9.78 184.87 ± 9.45 0.07 ALT5 (mg/L) 23.97 ± 9.50 27.21 ± 4.28 24.03 ± 6.37 22.99 ± 1.74 0.11 SOD6 (U/mL) 174.96 ± 7.37a 183.09 ± 6.51a 162.96 ± 6.86a 255.42 ± 6.86b 0.03 CHE7 (U/100 L) 81.97 ± 7.86 88.14 ± 6.01 80.02 ± 4.82 75.45 ± 3.71 0.08 ALKP8 (U/L) 75.21 ± 6.71 62.86 ± 8.96 78.78 ± 8.10 81.16 ± 9.12 0.06 GSH-PX9(umol/L) 454.68 ± 9.12a 484.65 ± 10.34a 494.18 ± 10.10a 524.55 ± 12.75b 0.03 CAT10 (U/L) 0.45 ± 0.01a 0.48 ± 0.01a 0.50 ± 0.01a 0.52 ± 0.02b 0.02 Note: values are mean ± SE in each group. Values not sharing a common superscript differ significantly at P < 0.05. EP0, EP 1, EP 2, EP3 indicates diets supplemented with 0, 1%, 3%, or 5% earthworm powder, respectively. 1Total protein. 2Albumin. 3Gamma-globulin. 4Glutamic oxaloacetic transaminase. 5Alanine aminotransferase. 6Superoxide dismutase. 7Cholinesterase. 8Alkaline phosphatase. 9Glutathione peroxidase. 10Catalase. View Large Table 4. Blood characteristics of broiler pullets fed earthworm powder. Parameters EP0 EP1 EP2 EP3 P-value TP1 (g/L) 56.26 ± 8.38a 66.59 ± 7.63b 62.81 ± 7.61b 66.79 ± 7.36b 0.03 ALB2 (g/L) 13.12 ± 1.09 14.42 ± 1.60 15.09 ± 2.46 13.01 ± 2.24 0.09 GLB3 (g/L) 43.14 ± 5.51a 52.17 ± 8.20a 47.72 ±6. 53a 53.78 ± 6.33b 0.04 GOT4 (U/L) 203.86 ± 8.15 213.35 ± 8.15 215.21 ± 9.78 184.87 ± 9.45 0.07 ALT5 (mg/L) 23.97 ± 9.50 27.21 ± 4.28 24.03 ± 6.37 22.99 ± 1.74 0.11 SOD6 (U/mL) 174.96 ± 7.37a 183.09 ± 6.51a 162.96 ± 6.86a 255.42 ± 6.86b 0.03 CHE7 (U/100 L) 81.97 ± 7.86 88.14 ± 6.01 80.02 ± 4.82 75.45 ± 3.71 0.08 ALKP8 (U/L) 75.21 ± 6.71 62.86 ± 8.96 78.78 ± 8.10 81.16 ± 9.12 0.06 GSH-PX9(umol/L) 454.68 ± 9.12a 484.65 ± 10.34a 494.18 ± 10.10a 524.55 ± 12.75b 0.03 CAT10 (U/L) 0.45 ± 0.01a 0.48 ± 0.01a 0.50 ± 0.01a 0.52 ± 0.02b 0.02 Parameters EP0 EP1 EP2 EP3 P-value TP1 (g/L) 56.26 ± 8.38a 66.59 ± 7.63b 62.81 ± 7.61b 66.79 ± 7.36b 0.03 ALB2 (g/L) 13.12 ± 1.09 14.42 ± 1.60 15.09 ± 2.46 13.01 ± 2.24 0.09 GLB3 (g/L) 43.14 ± 5.51a 52.17 ± 8.20a 47.72 ±6. 53a 53.78 ± 6.33b 0.04 GOT4 (U/L) 203.86 ± 8.15 213.35 ± 8.15 215.21 ± 9.78 184.87 ± 9.45 0.07 ALT5 (mg/L) 23.97 ± 9.50 27.21 ± 4.28 24.03 ± 6.37 22.99 ± 1.74 0.11 SOD6 (U/mL) 174.96 ± 7.37a 183.09 ± 6.51a 162.96 ± 6.86a 255.42 ± 6.86b 0.03 CHE7 (U/100 L) 81.97 ± 7.86 88.14 ± 6.01 80.02 ± 4.82 75.45 ± 3.71 0.08 ALKP8 (U/L) 75.21 ± 6.71 62.86 ± 8.96 78.78 ± 8.10 81.16 ± 9.12 0.06 GSH-PX9(umol/L) 454.68 ± 9.12a 484.65 ± 10.34a 494.18 ± 10.10a 524.55 ± 12.75b 0.03 CAT10 (U/L) 0.45 ± 0.01a 0.48 ± 0.01a 0.50 ± 0.01a 0.52 ± 0.02b 0.02 Note: values are mean ± SE in each group. Values not sharing a common superscript differ significantly at P < 0.05. EP0, EP 1, EP 2, EP3 indicates diets supplemented with 0, 1%, 3%, or 5% earthworm powder, respectively. 1Total protein. 2Albumin. 3Gamma-globulin. 4Glutamic oxaloacetic transaminase. 5Alanine aminotransferase. 6Superoxide dismutase. 7Cholinesterase. 8Alkaline phosphatase. 9Glutathione peroxidase. 10Catalase. View Large Antioxidant enzymes (SOD, CAT, and GSH-Px) protect the body from oxidative damage [25]. Superoxide dismutase and CAT can eliminate free radicals during xenobiotic exposure [29]. Glutathione peroxidase is a peroxide decomposition enzyme, which uses glutathione as a substrate to transform H2O2 into H2O [30]. Glutathione is a major nonprotein thiol in living organisms and plays a central role in coordinating the body's antioxidant defense mechanism. Prakash et al. [15] reported that earthworm powder exhibits potent antioxidant and hepatoprotective properties in alcohol-hepatotoxic rats. The present study showed that a diet containing 5% earthworm powder increased GLB, SOD, GSH-Px, and CAT activities in the pullets. These findings indicate that dietary earthworm powder supplementation can increase the antioxidant capacity of the body, and supplementing broiler diets with earthworm powder might improve the bird's antioxidant system. Nonsignificant differences were observed in the levels of ALB, GOT, GPT, and ALKP in all the treatment groups. The differences between the days 1 and 30 were not statistically significant with respect to any of the parameters we measured. Heavy Metal Residues The levels of the heavy metal residues in the chicken thigh muscles, chest muscles, and livers are shown in Table 5. The values measured in this study were compared with China's permissible limits given in the “General Standard of Contaminants in Foods” (GB 2762–2012). All the samples collected from the livers showed higher Cu, Zn, Pb, Cr, Cd, and As levels than the samples collected from both the groups of muscles. Table 5. Heavy metal residues of broiler pullets fed earthworm powder. Groups Item Species EP0 EP1 EP2 EP3 P-value Cu (mg/kg) Chest muscle 2.60 ± 0.39 2.40 ± 0.24 2.30 ± 0.72 2.51 ± 0.48 0.17 Thigh muscle 2.39 ± 0.41 2.47 ± 0.28 2.31 ± 0.37 2.45 ± 0.26 0.45 Liver 14.13 ± 1.63b 8.82 ± 1.57a 8.98 ± 1.67a 8.52 ± 1.83a 0.04 Zn (mg/kg) Chest muscle 16.79 ± 2.24 17.07 ± 3.11 15.30 ± 2.04 21.16 ± 3.87 0.06 Thigh muscle 57.91 ± 5.21 61.75 ± 3.62 65.97 ± 4.02 65.08 ± 3.97 0.06 Liver 94.63 ± 3.25 96.87 ± 2.81 97.84 ± 5.58 103.91 ± 6.73 0.09 Pb (mg/kg) Chest muscle 0.09 ± 0.02 0.09 ± 0.03 0.08 ± 0.03 0.09 ± 0.01 0.71 Thigh muscle ND ND ND ND Liver 0.42 ± 0.22 0.38 ± 0.13 0.45 ± 0.12 0.48 ± 0.22 0.23 Cr (μg/kg) Chest muscle 37.34 ± 6.03 44.56 ± 1.32 52.51 ± 2.55 49.60 ± 1.99 0.08 Thigh muscle 41.24 ± 3.61 56.84 ± 3.27 61.92 ± 4.52 45.57 ± 1.92 0.06 Liver 60.17 ± 2.16 68.61 ± 5.17 62.57 ± 1.44 58.74 ± 2.78 0.15 Cd (μg/kg) Chest muscle 16.65 ± 3.68 16.87 ± 4.14 12.58 ± 2.42 16.81 ± 1.62 0.21 Thigh muscle 12.80 ± 7.32 19.24 ± 4.61 ND ND 0.07 Liver 111.10 ± 8.03 142.40 ± 9.67 125.41 ± 4.23 183.53 ± 4.44 0.38 As (mg/kg) Chest muscle 0.21 ± 0.06 0.18 ± 0.03 0.22 ± 0.04 0.22 ± 0.03 0.36 Thigh muscle 0.28 ± 0.06a 0.24 ± 0.02a 0.34 ± 0.08b 0.26 ± 0.08a 0.02 Liver 2.52 ± 0.41 2.77 ± 0.47 4.76 ± 0.45 4.11 ± 1.18 0.07 Groups Item Species EP0 EP1 EP2 EP3 P-value Cu (mg/kg) Chest muscle 2.60 ± 0.39 2.40 ± 0.24 2.30 ± 0.72 2.51 ± 0.48 0.17 Thigh muscle 2.39 ± 0.41 2.47 ± 0.28 2.31 ± 0.37 2.45 ± 0.26 0.45 Liver 14.13 ± 1.63b 8.82 ± 1.57a 8.98 ± 1.67a 8.52 ± 1.83a 0.04 Zn (mg/kg) Chest muscle 16.79 ± 2.24 17.07 ± 3.11 15.30 ± 2.04 21.16 ± 3.87 0.06 Thigh muscle 57.91 ± 5.21 61.75 ± 3.62 65.97 ± 4.02 65.08 ± 3.97 0.06 Liver 94.63 ± 3.25 96.87 ± 2.81 97.84 ± 5.58 103.91 ± 6.73 0.09 Pb (mg/kg) Chest muscle 0.09 ± 0.02 0.09 ± 0.03 0.08 ± 0.03 0.09 ± 0.01 0.71 Thigh muscle ND ND ND ND Liver 0.42 ± 0.22 0.38 ± 0.13 0.45 ± 0.12 0.48 ± 0.22 0.23 Cr (μg/kg) Chest muscle 37.34 ± 6.03 44.56 ± 1.32 52.51 ± 2.55 49.60 ± 1.99 0.08 Thigh muscle 41.24 ± 3.61 56.84 ± 3.27 61.92 ± 4.52 45.57 ± 1.92 0.06 Liver 60.17 ± 2.16 68.61 ± 5.17 62.57 ± 1.44 58.74 ± 2.78 0.15 Cd (μg/kg) Chest muscle 16.65 ± 3.68 16.87 ± 4.14 12.58 ± 2.42 16.81 ± 1.62 0.21 Thigh muscle 12.80 ± 7.32 19.24 ± 4.61 ND ND 0.07 Liver 111.10 ± 8.03 142.40 ± 9.67 125.41 ± 4.23 183.53 ± 4.44 0.38 As (mg/kg) Chest muscle 0.21 ± 0.06 0.18 ± 0.03 0.22 ± 0.04 0.22 ± 0.03 0.36 Thigh muscle 0.28 ± 0.06a 0.24 ± 0.02a 0.34 ± 0.08b 0.26 ± 0.08a 0.02 Liver 2.52 ± 0.41 2.77 ± 0.47 4.76 ± 0.45 4.11 ± 1.18 0.07 Note: values are mean ± SE in each group. Values not sharing a common superscript differ significantly at P < 0.05. “ND” means no detection. EP0, EP 1, EP 2, and E3 indicates diets supplemented with 0, 1%, 3%, or 5% earthworm powder, respectively. View Large Table 5. Heavy metal residues of broiler pullets fed earthworm powder. Groups Item Species EP0 EP1 EP2 EP3 P-value Cu (mg/kg) Chest muscle 2.60 ± 0.39 2.40 ± 0.24 2.30 ± 0.72 2.51 ± 0.48 0.17 Thigh muscle 2.39 ± 0.41 2.47 ± 0.28 2.31 ± 0.37 2.45 ± 0.26 0.45 Liver 14.13 ± 1.63b 8.82 ± 1.57a 8.98 ± 1.67a 8.52 ± 1.83a 0.04 Zn (mg/kg) Chest muscle 16.79 ± 2.24 17.07 ± 3.11 15.30 ± 2.04 21.16 ± 3.87 0.06 Thigh muscle 57.91 ± 5.21 61.75 ± 3.62 65.97 ± 4.02 65.08 ± 3.97 0.06 Liver 94.63 ± 3.25 96.87 ± 2.81 97.84 ± 5.58 103.91 ± 6.73 0.09 Pb (mg/kg) Chest muscle 0.09 ± 0.02 0.09 ± 0.03 0.08 ± 0.03 0.09 ± 0.01 0.71 Thigh muscle ND ND ND ND Liver 0.42 ± 0.22 0.38 ± 0.13 0.45 ± 0.12 0.48 ± 0.22 0.23 Cr (μg/kg) Chest muscle 37.34 ± 6.03 44.56 ± 1.32 52.51 ± 2.55 49.60 ± 1.99 0.08 Thigh muscle 41.24 ± 3.61 56.84 ± 3.27 61.92 ± 4.52 45.57 ± 1.92 0.06 Liver 60.17 ± 2.16 68.61 ± 5.17 62.57 ± 1.44 58.74 ± 2.78 0.15 Cd (μg/kg) Chest muscle 16.65 ± 3.68 16.87 ± 4.14 12.58 ± 2.42 16.81 ± 1.62 0.21 Thigh muscle 12.80 ± 7.32 19.24 ± 4.61 ND ND 0.07 Liver 111.10 ± 8.03 142.40 ± 9.67 125.41 ± 4.23 183.53 ± 4.44 0.38 As (mg/kg) Chest muscle 0.21 ± 0.06 0.18 ± 0.03 0.22 ± 0.04 0.22 ± 0.03 0.36 Thigh muscle 0.28 ± 0.06a 0.24 ± 0.02a 0.34 ± 0.08b 0.26 ± 0.08a 0.02 Liver 2.52 ± 0.41 2.77 ± 0.47 4.76 ± 0.45 4.11 ± 1.18 0.07 Groups Item Species EP0 EP1 EP2 EP3 P-value Cu (mg/kg) Chest muscle 2.60 ± 0.39 2.40 ± 0.24 2.30 ± 0.72 2.51 ± 0.48 0.17 Thigh muscle 2.39 ± 0.41 2.47 ± 0.28 2.31 ± 0.37 2.45 ± 0.26 0.45 Liver 14.13 ± 1.63b 8.82 ± 1.57a 8.98 ± 1.67a 8.52 ± 1.83a 0.04 Zn (mg/kg) Chest muscle 16.79 ± 2.24 17.07 ± 3.11 15.30 ± 2.04 21.16 ± 3.87 0.06 Thigh muscle 57.91 ± 5.21 61.75 ± 3.62 65.97 ± 4.02 65.08 ± 3.97 0.06 Liver 94.63 ± 3.25 96.87 ± 2.81 97.84 ± 5.58 103.91 ± 6.73 0.09 Pb (mg/kg) Chest muscle 0.09 ± 0.02 0.09 ± 0.03 0.08 ± 0.03 0.09 ± 0.01 0.71 Thigh muscle ND ND ND ND Liver 0.42 ± 0.22 0.38 ± 0.13 0.45 ± 0.12 0.48 ± 0.22 0.23 Cr (μg/kg) Chest muscle 37.34 ± 6.03 44.56 ± 1.32 52.51 ± 2.55 49.60 ± 1.99 0.08 Thigh muscle 41.24 ± 3.61 56.84 ± 3.27 61.92 ± 4.52 45.57 ± 1.92 0.06 Liver 60.17 ± 2.16 68.61 ± 5.17 62.57 ± 1.44 58.74 ± 2.78 0.15 Cd (μg/kg) Chest muscle 16.65 ± 3.68 16.87 ± 4.14 12.58 ± 2.42 16.81 ± 1.62 0.21 Thigh muscle 12.80 ± 7.32 19.24 ± 4.61 ND ND 0.07 Liver 111.10 ± 8.03 142.40 ± 9.67 125.41 ± 4.23 183.53 ± 4.44 0.38 As (mg/kg) Chest muscle 0.21 ± 0.06 0.18 ± 0.03 0.22 ± 0.04 0.22 ± 0.03 0.36 Thigh muscle 0.28 ± 0.06a 0.24 ± 0.02a 0.34 ± 0.08b 0.26 ± 0.08a 0.02 Liver 2.52 ± 0.41 2.77 ± 0.47 4.76 ± 0.45 4.11 ± 1.18 0.07 Note: values are mean ± SE in each group. Values not sharing a common superscript differ significantly at P < 0.05. “ND” means no detection. EP0, EP 1, EP 2, and E3 indicates diets supplemented with 0, 1%, 3%, or 5% earthworm powder, respectively. View Large There was no significant difference in the Cu levels between the samples collected from the thigh and chest muscles of groups EP1, EP2, and EP3. The concentrations of Cu in the liver samples exhibited significant differences (P = 0.04) between EP0 and the other groups. This difference might have been caused by the increase in the SOD level with the addition of earthworm powder. Because Cu is an important component of SOD [31], as the SOD level increased, the Cu level in the liver decreased correspondingly to form Cu-SOD. The Cu levels in all the samples were below China's permissible limit. The mean Zn levels in each tissue type showed no significant differences among the treatment groups (P > 0.05). The highest Zn levels of 94.63 ± 3.25, 96.87 ± 2.81, 97.84 ± 5.58, and 103.91 ± 6.73 mg kg−1 were all detected in the liver samples, which indicated that the liver is possibly the main Zn accumulation area. All the values in the studied samples were below China's permissible limit. There were nonsignificant differences in the levels of Pb, Cr, and Cd among all the treatment groups, with the concentrations of all the 3 heavy metals generally <0.5 mg kg−1 in the samples from all the groups. The levels found in this study were much lower than China's permissible limits of 1 mg/kg. However, there were relatively high levels of As (2.52 ± 0.41, 2.77 ± 0.47, 4.76 ± 0.45, and 4.11 ± 1.18 mg/kg in EP0, EP1, EP2, and EP3, respectively) in the liver. There is no permissible limit for As listed in the National Food Safety Standard GB 2762–2012 of the People's Republic of China. In general, the liver contained higher levels of heavy metals than did the thigh and chest muscles. All the heavy metal residues in the thigh muscle, chest muscle, and liver were below China's permissible limits. CONCLUSIONS AND APPLICATIONS Dietary earthworm powder supplementation can increase the antioxidant capacity of the body, and supplementation of broiler diets with earthworm powder might improve the antioxidant system of the broilers. Heavy metals in poultry diets are maintained below the permitted levels based on the National Feed Hygiene Standard GB 13078–2001 of the People's Republic of China. The residues of all the heavy metals in the thigh, chest muscles, and liver of the broiler pullets, following supplementation of the broiler diet with earthworm powder, were below China's permissible limits. Therefore, earthworm powder can be used as a feed supplement in broiler diets. Note Primary Audience: Nutritionists, Quality Assurance Personnel, Researchers, Veterinarians REFERENCES AND NOTES 1. Ravindran V. , Blair R. . 1993 . 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Pharmacokinetic and anti-inflammatory properties in the rat of superoxide dismutases (Cu SODs and Mn SOD) from various species . Biochem. Pharmacol. 33 : 2755 – 2760 . Acknowledgements This work was supported by the Fund for National science and technology support plan (2012BAD14B14), Science and Technology Plan Projects of Department of Education of Jiangxi Province (grant number: GJJ160401). © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Journal of Applied Poultry ResearchOxford University Press

Published: Dec 1, 2018

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