Effects of replacing inorganic trace minerals with organic trace minerals on the production performance, blood profiles, and antioxidant status of broiler breeders

Effects of replacing inorganic trace minerals with organic trace minerals on the production performance, blood profiles, and antioxidant status of broiler breeders

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College of Animal Science, Zhejiang University, Hangzhou 310058, China; † Alltech Biological Products (China) Co., Ltd. Beijing 101400, China; and ‡ Center for Animal Nutrigenomics and Applied Animal Nutrition, Alltech Inc, Nicholasville, KY 40356, USA or L-OTM treatments. OTM and COM treatments both had increased serum LH and P4 . The relatively higher mineral levels fed in COM and OTM treatments increased blood total protein (P < 0.05). In addition, activities of serum GSH-Px, Mn-SOD, and T-SOD were higher (P < 0.01), while malondialdehyde (MDA) content was lower (P < 0.05), for COM and OTM birds as compared to L-ITM and VL-OTM. The serum T-SOD of L-OTM birds was significantly higher (9.81%; P < 0.01) than that of L-ITM birds. Higher (P < 0.05) activities of liver GSH-Px and T-SOD, and lower MDA concentrations (P < 0.01) were measured in the COM, L-OTM, and OTM treatments than the L-ITM treatment. Collectively, total replacement of high levels of ITMs by lower levels of OTMs in broiler breeder diets was beneficial for productive performance under the conditions of this study.

ABSTRACT This study was conducted to investigate the effects of replacing inorganic trace minerals (ITMs) with organic trace minerals (OTMs) on the production performance, blood profile, and antioxidative status of broiler breeders. A total of 600 healthy broiler breeder hens, aged 40 wk, were randomly divided into 5 treatments with 4 replicates in each treatment, and fed for 10 wk. Experimental treatments were: (1) commercial levels of inorganic minerals (COM); (2) L-ITM (50% of the COM, except for Se); (3) VL-OTM (37.5% of the COM, except for Se); (4) L-OTM (equivalent to L-ITM); and (5) OTM (62.5% of the COM, except for Se). The laying rate was 9.56% higher, feed-to-egg ratio was 7.83% lower, and rate of qualified eggs was 18.33% higher (P < 0.05) for L-OTM compared to L-ITM despite equal mineral levels. The fertility with COM was significantly higher (P < 0.05) than L-ITM, VL-OTM,

Key words: organically bound trace mineral, broiler breeder, production performance, blood profile, antioxidant status 2019 Poultry Science 0:1–8 http://dx.doi.org/10.3382/ps/pez035

INTRODUCTION

Although, mineral requirements are lower than other nutrients, antagonism between minerals and interaction with feed components can reduce bioavailability and frequently result in deficiencies. The effects of Zn, Mn, and Cu deficiencies on breeder performance and embryo development have been well documented and can result in low egg production, poor fertility and hatchability, or even reductions in eggshell and bone strength (Favero et al., 2013). Typically, large safety margins for minerals levels are used in feed formulation (Bao et al., 2007) to meet the nutrition requirement, owing to inexpensive costs and wide materials sources (oxides and sulfates). Excessive use of ITMs can result in a detrimental effect on the environment because some of the excess minerals are not assimilated and are excreted with the feces (Lukasz et al., 2017). Enhancing mineral utilization is one of the most effective measurements to ensure both the animal healthy and reduction of the environmental burden.

Trace minerals (TMs) play vital roles in the production of broiler breeder, not only in health maintenance, but also the quality of eggs and even offspring (Dibner et al., 2007; M’Sadeq et al., 2018). TMs are necessary for proper functioning of various digestive, physiological, and biosynthetic processes and as cofactors of many metalloenzymes, including the Zn/Cu-SOD, Mn-SOD, and Se-containing enzyme such as GSH-Px (Echeverry et al., 2016). TMs are also constituents of hundreds of proteins involved in intermediary metabolism, hormone secretion, and immune function (Dieck et al., 2003).

 C 2019 Poultry Science Association Inc. Received April 19, 2018. Accepted January 23, 2019. 1 Corresponding author: [email protected] This research is appropriate for Physiology and Reproduction.

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G. Wang,∗ L. J. Liu,∗ W. J. Tao,∗ Z. P Xiao,∗ X. Pei,∗ B. J. Liu,∗ M. Q. Wang,∗,1 G. Lin,† and T. Y. Ao‡

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MATERIALS AND METHODS Experimental Animals All experimental procedures were conducted in accordance with the Animal Welfare Committee guidelines and approved by the Animal Science College of Zhejiang University (Hangzhou, China). A total of 600 healthy “ZhenNing” yellow feather broiler breeders (age: 40 wk; BW: 1.70 ± 0.07 kg) were randomly divided into 5 treatments, with 4 replicates in each treatment, 30 hens per replicate, and 3 hens per cage (cage parameters 50 cm × 50 cm × 50 cm). The experiment lasted for 10 wk (including 2 wk for adaptation). Ambient conditions such as temperature, lighting, and humidity were conducted according to standard farm management practices; broiler breeders were allowed ad libitum access to feed mixtures, which was appropriate for laying hens. Artificial insemination was carried out every 5 d to maintain the fertilization.

Dietary Treatments The basal diet (Table 1) was formulated according to the recommendation for broiler breeder by NRC (1994) and modified with NY/T 33-2004 (2004). The supplemental doses of mineral premix are presented in Table 2 with 2 ITMs treatments and 3 OTMs treatments. (1) Commercially recommended levels of ITMs (30 mg Fe as FeSO4 , 80 mg Zn as ZnSO4 , 80 mg Mn as MnSO4 , 10 mg Cu as CuSO4 , 0.3 mg Se as sodium selenite, per kilogram of diet; COM); (2) L-ITM (match to 50% of the COM, 15 mg Fe as FeSO4 , 40 mg Zn as ZnSO4 , 40 mg Mn as MnSO4 , 5 mg Cu as CuSO4 , except for 0.3 mg Se as sodium selenite, per kilogram of diet); (3) VL-OTM (37.5% of the COM, except for 0.3 mg Se per kilogram of diet); (4) L-OTM (equivalent to L-ITM); (5) OTM (62.5% of the COM, except for 0.375 mg Se per kilogram of diet). The organic trace elements Zn, Fe, Mn, and Cu were provided as mineral proteinates

Table 1. Composition and nutrient levels of the basal diets (as feed basis). Nutrient levelsb

Ingredients (%) Corn Soybean meal Fish meal Wheat bran Soybean oil Shell power Limestone CaHPO4 NaCl NaHCO3 DL-Met Choline chloride (50%) Vitamin and mineral premixa Total

58.3 22 3 4 0.7 8 1.6 0.7 0.2 0.3 0.1 0.1 1 100

ME (MJ/kg) CP Lys Met Ca TP

10.92 18.25 0.95 0.41 3.73 0.56

a Provided per kilogram of diet: Vitamin A 10,800 IU, Vitamin D3 2160 IU, Vitamin E 20 IU, Vitamin K3 1.4 mg, Vitamin B1 1.8 mg, Vitamin B2 8 mg, Vitamin B6 4.1 mg, Vitamin B12 0.01 mg, pantothenate 11 mg, nicotinic acid 32 mg, folic acid 1.1 mg, biotin 0.18 mg, phytase 1000 IU. The TMs supplementation were referred to our experimental design. b ME based on calculated values; others were analyzed values.

(protein ratio ranging from 21% to 33%, analyzed data), with Se as organic selenium yeast (Bioplex PP, Alltech Inc., Nicholasville, KY). TM content of the basal diet was measured using an atomic absorption spectrometer (ICE-3500, Thermo Crop., USA) for Cu, Zn, Fe, Mn, and an atomic fluorescence spectrometer (AFS-230E, HaiGuang Crop., Beijing, China) for Se following microwave wet digestion with nitric acid.

Samples Collection and Measurement Laying Performance. Weight and number of qualified eggs were recorded daily for each replicate, feed consumption was recorded weekly by replicate and mortality was recorded throughout the experiment. Laying rate, feed-to-egg ratio (kilogram of feed/kilogram of egg), and daily feed intake (g/bird per day) were calculated at the end of the trial. Reproductive Performance and Egg Quality. 20 qualified eggs from each replicate (total 400) were collected during the 3rd, 5th, and 7th week of production with treatment diets to measure fertility and hatchability. Blood was collected from the wing vein of 3 broilers per replicate using a syringe and used to determine serum luteinizing hormone (LH), estradiol (E2 ), and progesterone (P4 ) by using an Automatic Biochemical Analyzer (AU5421, Olympus Crop., Japan). At the 8th week of production with the treatment diets, six eggs from each replicate were collected (24 eggs per treatment) for egg quality measurement using a digital egg tester (DET-6000, Nabel Co., Ltd, Kyoto, Japan) according to the previously reported methods of Xiao et al. (2014) and Yilmaz et al. (2015). Blood Profiles and Antioxidant Status. At the end of the trial, 3 hens of each replicate (total 60) were selected to collect the serum from the wing vein following centrifuged at 3,000 rpm for 15 min to get the serum and stored in tubes at −20◦ C for analysis.

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Compared to the corresponding source of inorganic mineral, forms of amino acid chelates or proteinates are characterized by better chemical stability and physical heterogeneity and are better absorbed in gut due to reduced interaction with other ration components (Stefanello et al., 2014). Studies have reported that organically bound TMs may be absorbed via amino acid or peptide transport pathways, more effectively than through mineral pathways, which could explain their higher utilization (Muszy´ nski et al., 2018). However, data on complete replacement of ITMs by corresponding organic trace minerals (OTMs) at certain levels in broiler breeder diets are limited. Therefore, our study was conducted to investigate the effects of total replacement of ITMs by recommended levels of OTMs on laying and reproductive performance, blood profiles, tissue mineral contents, and antioxidant status in broiler breeders.

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ORGANIC TRACE MINERAL AND BROILER BREEDER Table 2. Assayed mineral content of broiler breeder diets (mg/kg). Inorganic treatmentsa Item

110.8 186.9 106.3 18.1 0.4

(80)c (30) (80) (10) (0.300)

L-ITM 68.8 172.0 56.3 12.5 0.4

(40) (15) (40) (5) (0.300)

VL-OTM 58.8 165.0 46.3 11.0 0.3

(30) (11.25) (30) (3.75) (0.225)

L-OTM 65.8 169.0 57.5 12.3 0.4

(40) (15) (40) (5) (0.300)

OTM 78.8 175.1 72.5 13.5 0.5

(50) (18.75) (50) (6.25) (0.375)

a Inorganic treatments include COM and L-ITM treatments in which the minerals were provided by the sulfates of Zn, Fe, Mn, Cu and sodium selenite for Se. b Organic treatments include VL-OTM, L-OTM, and OTM treatments in which the minerals were provided by the Bioplex PP, Alltech Inc (Beijing, China). Proteinates of Zn, Fe, Mn, Cu, and selenium yeast for Se. c () Supplemental mineral.

Alkaline phosphatase (ALP), total protein (TP), albumin (ALB), uric acid (UA), serum calcium (Ca), phosphorus (P), and glucose (Glu) were analyzed using an Automatic Biochemical Analyzer (AU5421, Olympus Crop., Japan). Ceruloplasmin and hemoglobin were analyzed using ELISA kits (Jining Bioengineering Institute, Shanghai, China). Livers were removed and washed with cold PBS immediately following stored at the liquid nitrogen until analysis. Antioxidant enzymes containing glutathione peroxidase (GSH-Px), total superoxide dismutase (TSOD), Cu/Zn superoxide dismutase (Cu/Zn-SOD), Mn superoxide dismutase (Mn-SOD) activity, and concentration of malondialdehyde (MDA) in liver and serum samples were determined according to the instructions of corresponding kits purchased from Jiancheng Bioengineering Institute (Nanjing, China). Total protein in liver was also measured by Enhanced BCA Protein Assay Kit purchased from the Beyotime Biotechnology (Beijing, China).

whole experimental period. When the average laying rates for the entire study were calculated, the rates of L-OTM birds outperformed the commercial levels of inorganic minerals (COM) treatment (P < 0.05). Similar trends were observed for feed-to-egg ratio, where the ratio was consistently lower (P < 0.05) for L-OTM in comparison with other treatments.

Reproductive Performance The results of fertility and hatchability are shown in Figure 2. COM birds showed better reproductive performance, where the fertility was higher than the other treatments; this result was similar when measured for the entirety of the study (P < 0.05). Lower hatchability was noted in the VL-OTM treatment compared to the other treatments (P < 0.05), with no difference among the other treatments (P > 0.05). Regarding gonadal hormones, L-ITM and VL-OTM resulted in lower serum LH and P4 (P < 0.05), no other differences were observed among all treatments (P > 0.05).

Statistical Analysis The data were analyzed using one-way ANOVA with the procedure of SPSS 20.0, and defined dietary treatment as the independent variable. One dietary treatment was served as the experimental unit, which contains four replicates, and one replicate was served as the statistical unit. Data are expressed as means ± SEM. The post hoc Tukey test was used to distinguish statistical significant differences between treatments. A significance level was set at P < 0.05.

RESULTS Laying Performance Data were calculated biweekly during the trial. No significant difference in feed intake among the treatments (Figure 1B) was noticed during the trial. However, different effects on the laying rate and feed-to-egg ratio were shown in Figures 1A and 1C. The laying rate of birds fed L-OTM was 9.56% higher than that of bird fed L-ITM with the equal mineral levels (P < 0.05) on the 7th to 8th week and over the

Egg Quality The effects of dietary treatment on egg quality were shown in Table 3. The main significant effects in this study were lower (P < 0.05) rate of qualified eggs and egg weight when birds were supplemented with L-ITM. With respect to the yolk color, the VL-OTM had poorer color that was based on the score, when compared to the other organic treatments (P < 0.05). Eggshell quality such as the eggshell thickness and strength, and egg inner quality including the albumin height and haugh unit showed the similar effects irrespective the mineral sources.

Blood Profiles The total protein, albumin, and glucose contents (Table 4) in serum were significantly lower in L-ITM birds than COM birds (P < 0.05), similarly, the VLOTM treatment had decreased the albumin compared to COM (P < 0.05). The total protein in serum was increased as OTMs levels increased. L-OTM and OTM

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Zn Fe Mn Cu Se

COM

Organic treatmentsb

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treatments showed similar effects on blood profiles compared with the COM treatment. Similar trends were observed for ceruloplasmin and hemoglobin.

Antioxidant Status Antioxidant enzyme activity in serum and liver of broiler were shown in Table 5. COM, L-OTM, and OTM feeding increased the activity of both GSH-Px and T-SOD in the serum (P < 0.05), which corresponded with a reduction in MDA levels. The blood samples from birds fed L-ITM and VL-OTM had lower (P < 0.01) activities of GSH-Px and T-SOD and higher (P < 0.05) MDA concentration compared to other treatments. Correspondingly, antioxidant status in liver was similar to serum. Livers from birds fed COM, LOTM, and OTM had higher (P < 0.01) GSH-Px and T-SOD enzyme activity and lower MDA concentration compared to livers from birds fed L-ITM and VL-OTM, this indicated the enhancement of antioxidant defense functions.

DISCUSSION In our study, there was no difference among birds fed with three levels of OTMs diets on production performances. However, only L-OTM increased the laying rate and reduced the ratio of feed to egg compared with the equivalent L-ITM, which indicated that the organic

forms of minerals had the potential to improve laying performance. However, other researchers have reported variable effects with the addition of organic minerals on egg production. Lim and Paik (2003) showed that the combination of organic Zn and Mn tended to decrease egg production, while the combination of organic Zn, Mn, and Cu did not affect egg production. Similar results were also observed by Swiatkiewicz and Koreleski (2008), who found that partial or complete substitution of inorganic Zn and Mn oxides with metal-amino acid complexes had no effects on laying performance parameters. On the other hand, Klecker et al. (2002) reported that substitution of 20% or 40% Mn and Zn from inorganic sources by their organic chelates significantly increased the laying performance of hens from 20 to 60 wk of age. The inconsistency in the behavioral responses of minerals among earlier reports may be due to the differences in diet composition, breed, the duration of the experiment, or other factors (Yenice et al., 2015). Feed intake had no significant difference among the diet treatments, this may be due to the use of mature broiler breeder in this study, as their primary organs and skeletons had been fully developed and the supplementation of minerals in the feed were sufficient to their requirements. However, a previous study (Leeson and Caston, 2008) indicated that lower levels of organic Fe, Cu, Zn, and Mn did not affect feed intake compared with inorganic forms in chickens, which means the mineral requirements were not only dependent on developmental degree but also the animal’s physiological status.

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Figure 1. Effects of different diets on production performance of the “ZhenNing” yellow feather broiler breeder. Four phases with 2 wk each. (A) Laying rate was calculated daily per replicate and the average of each 2 wk was used for statistic. (B) Feed intake measured on a weekly basis and was divided by the cumulative total numbers of the broilers within 1 wk. (C) Feed-to-egg ratio was calculated by dividing the egg weight by the feed consumption (kg/kg). All data were presented as mean ± SEM, n = 4. An asterisk ∗ indicates significant difference between two treatments at a level of P < 0.05.

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Table 3. Effect of different sources and supplemental levels of trace minerals on egg quality. Inorganic treatments Item Rate of qualified eggs (%) Egg weight (g) Yolk color (YCF) Eggshell strength (Kgf) Eggshell thickness (mm) Albumin height (mm) Haugh unit

Organic treatments

COM

L-ITM

VL-OTM

L-OTM

OTM

SEM

P-value

52.52a,b 50.15a 8.00a,b 4.00 0.38 4.64 69.51

45.00b 47.89b 8.00a,b 3.96 0.37 4.63 70.10

50.93a,b 48.86a,b 7.75b 4.18 0.37 4.55 69.16

53.25a 49.37a,b 8.25a 4.07 0.38 4.56 68.73

55.42a 49.67a,b 8.46a 4.44 0.38 4.56 68.32

5.87 0.32 0.08 0.09 0.004 0.06 0.55

0.018 0.049 0.021 0.748 0.624 0.991 0.897

Values within a row with no letter or different superscripts are significantly different (P < 0.05). Data are presented as mean ± SEM, n = 4.

Table 4. Effect of different sources and supplemental levels of trace minerals on the blood profiles. Inorganic treatments Item Total protein (g/L) Albumin (g/L) ALP (U/L) Ca (mmol/L) Glu (mmol/L) P (mmol/L) UA (nmol/L) Ceruloplasmin (U/L) Hemoglobin (U/L)

COM

Organic treatments

L-ITM a

69.41 20.18a 311.67 5.56 14.47a 1.80 224.86 76.07 179.66

b

61.66 18.25b 204.67 5.12 13.48b 1.47 206.51 59.90 133.32

VL-OTM a,b

64.16 18.41b 190.06 5.17 14.23a,b 1.61 204.48 64.41 137.33

L-OTM

OTM

SEM

P-value

a,b

a

1.01 0.27 26.83 0.10 0.14 0.07 7.79 2.88 8.86

0.027 0.044 0.659 0.565 0.026 0.729 0.933 0.071 0.163

66.24 19.19a,b 215.05 5.52 14.01a,b 1.64 216.17 69.30 155.53

68.73 19.58a,b 255.63 5.36 13.91a,b 1.67 219.08 76.62 189.60

Values within a row with no letter or different superscripts are significantly different (P < 0.05). Data are presented as mean ± SEM, n = 4.

The lowest fertility and hatchability of birds fed VL-OTM was observed in this study, which was reflected by the lowest blood concentration of gonadal hormones E2 , LH, and P4 that participate in regulating the follicle development and pregnancy maintenance (Sakumoto et al., 2014; Zabudskii, 2016). No more differences were found among others’ treatments with

an exception of the lower concentration of LH in L-ITM when compared with that in L-OTM. Zn, Cu, and Se are important for reproduction in males and females (Rajeswari and Swaminathan, 2014). Earlier reports had documented that OTMs improved the quantity of fertilized oocytes when compared to ITMs in Angus heifers, OTMs also benefited sow reproductive

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Figure 2. Effects of different diets on reproductive performance of the “ZhenNing” yellow feather broiler breeder. At the 3rd, 5th, and 7th week, 30 settable eggs were collected per replicate to determine fertility (A) and hatchability (B). (C, D, E) showed the serum estradiol (E2 ), luteinizing hormone (LH), progesterone (P4 ). Data were presented as mean ± SEM, n = 4. An asterisk ∗ indicates the significant difference between 2 different treatments where P < 0.05. ∗∗ indicates the significant level is at P < 0.01.

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Table 5. Effect of different sources and supplemental levels of trace minerals on antioxidant status in serum and liver. Inorganic treatments Item

2,493.03a 381.32a 298.63 82.69a 3.39b 62.67a 228.72a,b 199.63a 41.21 0.79b

Organic treatments

L-ITM

VL-OTM

L-OTM

OTM

SEM

P-value

1,900.62b 335.23b 287.08 48.14b 5.77a

1,907.91b 344.96b 283.03 61.93a,b 4.80a,b

2,598.63a 368.10a 294.78 73.32a,b 4.15a,b

2,573.63a 387.38a 300.18 87.20a 3.39b

91.12 5.31 6.93 5.52 0.32

0.001 0.001 0.803 0.045 0.008

34.72c 169.91c 152.74b 26.15 1.40a

39.88b 203.32b 175.91a,b 34.57 1.15a

47.66a,b 208.31b 190.59a,b 40.19 0.98b

67.74a 254.86a 200.09a 54.77 0.87b

3.31 7.47 6.93 4.74 0.06

0.009 < 0.001 0.012 0.451 0.001

Values within a row with no letter or different superscripts are significantly different (P < 0.05). Data are presented as mean ± SEM, n = 4.

performance (Lamb et al., 2008; Peters and Mahan, 2008). Organic minerals regulate the nutritionrelated factors affecting the quantity and quality of oocytes more efficiently than their inorganic counterparts (Lamb et al., 2008). Minerals are also capable of influencing hormonal secretions (Peters and Mahan, 2008), for example Cu plays an important role in the synthesis of LH by prostaglandin E2 (PGE2 ) and maintains the concentration of LH and folliclestimulating hormone (FSH) in serum (Rajeswari and Swaminathan, 2014; Sakumoto et al., 2014). While zinc finger proteins are involved in the genetic expression of gonadal hormone receptors (Tapiero and Tew, 2003). Research has also reported that Mn regulated the metabolism of cholesterol, which is the precursor of steroid hormones (Xie et al., 2014), and acts as the cofactor of mevalonate kinase and farnesyl pyrophosphate synthase. Previous studies in layer hens demonstrated the adverse effects of Mn deficiency on hormones such as the decrease of circulating progesterone, estradiol, LH and FSH (Feng and Feng, 1998; Yang, 2008). Works (Pine et al., 2005; Prestifilippo et al., 2008) in both male and female rats indicated that LH, FSH, and E2 increased while injecting MnCl2 into the brain. From our study, it seems that dietary inclusion of 30 mg/kg Mn in VL-OTM treatments was not enough for our broiler breeders to reach reproductive potential. Overall, the COM, L-OTM, and OTM birds showed no significant differences on egg quality, and the other 2 treatments showed worse performances on egg weight, yolk color, or rate of qualified eggs when compared with COM, L-OTM, and OTM treatments. TMs are vital for the eggshell formation (Fernandes et al., 2008). Zn and Mn are involved in formation of the organic matrix of the eggshell; and Cu plays a vital role in synthesis collagen that makes up the eggshell membrane (Stefanello et al., 2014). Here experimental data showed that egg weight was closely related to the supplemental dose, which was agreed with previous research where lower levels of mineral supplementation decreased egg weight in old hens (62 to 74 wk) but not in young hens (30 to 40 wk; Gheisari et al., 2010), this indicated that lay-

ing performance depended on the lifecycle phase. Other researchers have also stated that the substitution rate of inorganic minerals with organic forms of the minerals (Swiatkiewicz and Koreleski, 2008) and the relative combination of different organic minerals (Lim and Paik, 2003) influenced egg quality. The redness of yolk from birds fed the diets with organic minerals showed a dose-dependent pattern. Blood biochemical parameters are critical indicators of animal nutritional status. Total protein, albumin, and glucose were higher in COM and OTM treatments compared to L-ITM treatment. And some numerical improvements were observed on specific blood parameters in the birds fed with L-OTM when compared to the L-ITM, for example the total protein, ceruloplasmin, and hemoglobin were higher 7.43%, 15.69%, and 16.66%, respectively. Meanwhile, both the L-OTM and OTM treatments exhibited the similar effects on blood profiles as compared with the COM (P > 0.05). Feng et al. (2010) found the identical concentration of total protein when feeding a diet with lower level of organic Zn compared to its sulfates, even though the latter was 4 times higher than its organic source. One possibility is that minerals play a major role in the body enzyme system, physiology, metabolism, and growth, and are necessary to promote protein synthesis (Berger, 2006), such as the Zn can improve the feed digestibility by following activating pancreatic secretions digestive enzymes (Sahin et al., 2005), which subsequently results in higher level of glucose in the blood. Accordingly, the albumin concentration was increased as mineral supplementation levels increased, this is not unexpected as the albumin is the main transporter of minerals in serum (Lu et al., 2008; Sitar et al., 2013). GSH-Px and SOD are involved in breaking up the damage process by the clearance of reactive oxygen species (ROS; Bai et al., 2017). Se, Zn, Cu, and Mn are the cofactors of these 2 antioxidant enzymes. Generally, MDA is produced following peroxidation of polyunsaturated fatty acids and is a good marker of lipid peroxidation (Samuel et al., 2017). GSH-Px and SOD are the main enzymes that scavenge lipid peroxidation

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Serum GSH-Px (U/ml) T-SOD (U/ml) Cu/Zn-SOD (U/ml) Mn-SOD (U/ml) MDA (nmol/ml) Liver GSH-Px (U/mg Prot) T-SOD (U/mg Prot) Cu/Zn-SOD (U/mg Prot) Mn-SOD (U/mg Prot) MDA (nmol/mg Prot)

COM

ORGANIC TRACE MINERAL AND BROILER BREEDER

CONCLUSION The replacement of commercial level of inorganic minerals by L-OTM and OTM diets with lower level of proteinate in broiler breeder diets had no negative effect on production and reproduction performance, tissue antioxidant enzyme activity as well as blood profile. Supplementing diet OTMs with 50% level of commercial recommended supplementation (i.e., L-OTM diet) is the optimum ratio under the conditions of this study.

ACKNOWLEDGMENTS This research is financially supported by the Science and Technology Key Projects of Zhejiang Province, China (No.2015C02022) and Three Agricultural and Six-Party Research Cooperation Project of Zhejiang Province, China (No. CTZB-F180706LWZ-SNY1). We acknowledge the great support from the Ningbo Zhenning Animal Husbandry Ltd. Company, which provided the broiler breeder hens and procedural details of managing this trial. The same thanks to the Youjin Tu, Junbao Jiang, Lei Lu (Ningbo Zhenning Animal Husbandry Ltd. Company, Zhejiang, China) for the technical support in sample collection. Authors’ Contributions: The main work in this research including most experiments as well as manuscript writing was done by G. Wang, and L. J. Liu assisted to conduct breeding trial. Samples were pretreated with the help of W. J. Tao, and Z. P Xiao. The egg quality was measured by G. Wang, and L. J. Liu. Blood biochemical parameters were performed by L. J. Liu and X. Pei. Antioxidant status was done by G. Wang, W. J. Tao, and Z. P Xiao. G. Wang and W. J. Tao, conducted the statistical analysis. G. Wang, G. Lin, and T. Y. Ao conducted the manuscript revision. Prof. M. Q. Wang is in charge of the whole project and manuscript revision. All the authors read and approved the final manuscript.

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products such as MDA. L-OTM and OTM diets elevated the concentration of serum GSH-Px, T-SOD, and Mn-SOD compared to L-ITM and no difference was observed when compared to COM. This may indicate that organic minerals supplemented at lower levels have better effects on antioxidant defense. This corresponded to a reduction in MDA levels in serum consistent with the research (Aksu et al., 2010), where the replacement of inorganic Cu, Zn, and Mn by the corresponding organic elements reduced the MDA levels in broiler. COM birds exhibited better antioxidant status when compared with L-ITM treatment; however, no significant differences were seen when compared with L-OTM and OTM treatments. Organic zinc and copper had been shown to enhance the synthesis of Zn/Cu-SOD (Sahin et al., 2005; Sun et al., 2012). Organic Se previously increased the GSH-Px activity and antioxidant status (Jiang et al., 2009; Zhang et al., 2014) that is similar to our study. Sun et al. (2012) indicated that when organic Cu, Zn, Se, and Mn replaced inorganic forms, the former showed higher Zn/Cu-SOD contents at 39 wk and GSH-Px contents at 35 wk. These results indicated that antioxidant enzymes activities are related closely to the supplemental levels and age of birds. Similarly, antioxidant status was improved in liver by gradual increase of Zn supplementation regardless of its forms (Zhang et al., 2017). Aksu et al. (2010) indicated that broilers fed with lower levels of organically complexed Cu, Zn, and Mn displayed no liver damage due to oxidative stress. The results of the current study indicate that lower dose of organically bound TMs could meet the requirements of broiler breeders to exert their antioxidant potential, as the antioxidant status in liver of the OTM-supplemented birds were comparable or even better than the ITMs-supplemented birds at both commercial and lower levels. Yang et al. (2002) reported that increased GSH-Px activity in broiler liver was accompanied with supplemental Zn up to 50 ppm with the same level of Se, and Dai et al. (1999) also found the positive interaction between Zn and Se on the GSH-Px in dairy cow. In our study, higher activity of GSH-Px was observed both in serum and liver from COM-treated birds than that of fed supplemental L-ITM dietary with half level of Cu, Zn, Fe, Mn, and same level of Se, which could be resulted from the interaction between Zn and Se on GSH-Px activity.

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