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Effect of differing amounts of zinc oxide supplementation on the antioxidant status and zinc metabolism in newborn dairy calves
Livestock Science 230 (2019) 103819
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Effect of differing amounts of zinc oxide supplementation on the antioxidant status and zinc metabolism in newborn dairy calves Jingya Wei#, Fengtao Ma#, Liyuan Hao, Qiang Shan, Peng Sun
T
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State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
ARTICLE INFO
ABSTRACT
Keywords: Zinc oxide Zinc metabolism Antioxidant status Dairy calf
This study aimed to evaluate the effects of various amounts of zinc oxide (ZnO) supplementation on growth performance, incidence of diarrhea, micronutrient contents in feces and serum, antioxidant indicators and the concentrations of key enzymes and proteins involved in zinc metabolism in dairy calves. Forty newborn Holstein dairy calves were assigned to one of five groups according to a randomized complete block design: a control group (without zinc supplementation), or groups that received ZnO supplementation at 25.79, 51.58, 103.16, and 154.74 mg/d, yielding an amount of zinc consumption of 20 (Zn-20), 40 (Zn-40), 80 (Zn-80), and 120 (Zn120) mg/d, respectively, during the experimental period. The experiment lasted for 14 days, and blood and fecal samples were collected at the end of the trial. Supplementation with differing amounts of ZnO did not influence the growth performance or incidence of diarrhea of the calves (P > 0.05). However, the concentrations of fecal zinc and iron linearly increased with the amount of ZnO supplementation (P < 0.01). Compared with the control group, fecal zinc content was greater when calves were supplemented with zinc at concentrations of 40 and 120 mg/d, but the iron content was greater in the Zn-120 group than in the other four groups (P < 0.05). The amount of zinc supplementation did not affect the serum concentrations of micronutrients, including calcium, copper, iron, magnesium and phosphorus, in the newborn calves (P > 0.05). While the serum zinc concentration increased quadratically as the amount of supplementation increased, peaking with 80 mg zinc/d supplementation (P < 0.05). Increasing doses of ZnO supplementation linearly elevated the concentrations of metallothionein and superoxide dismutase (P < 0.01), and quadratically enhanced that of alkaline phosphatase (P < 0.05), but linearly reduced that of malondialdehyde in serum (P < 0.05). This study demonstrates that dietary ZnO supplementation to yield 80 mg zinc/d improves zinc metabolism and antioxidant status of newborn calves during their early life.
1. Introduction Diarrhea is one of the most important health problems in young calves, especially in neonatal calves (Virtala et al., 1996; Mayer et al., 2012), which is considered one of the principal causes of financial loss for dairy farms (Pourliotis et al., 2012). Zinc, a trace element, has many biological functions, including the maintenance of epithelia barrier integrity, cell division, and a sufficient immune response (Liberato et al., 2015; Schulte et al., 2016). Importantly, it has also been shown that zinc is an effective anti-inflammatory and anti-diarrheal agent (Oteiza and Mackenzie, 2005; Hu et al., 2013; Bonaventura et al., 2015). As the most effective method of administering zinc, oral Zn supplementation has been used globally in the prevention and treatment of diarrhea in infants and children, and millions of lives have been
saved in recent decades as a result (Prasad, 2014; Liberato et al., 2015). Simultaneously, the role of zinc in modulating oxidative stress has also been recognized (Prasad et al., 2004). Previous studies show that zinc supplementation can correct the increased plasma oxidative stress markers (Prasad, 2008). Dresler et al. (2016) reported that zinc methionine (Zn-Met) increased serum SOD activity in weaned female calves. Saleh et al. (2018) found that Zn-Met enhanced plasma glutathione peroxidase (GSH-Px) concentration compared with control in broilers exposed to high ambient temperature. Li et al. (2019) demonstrated that Zn-Met at 60 mg/kg elevated GSH-Px activity in serum and the SOD and GSH-Px activity in liver of laying hens. For decades, zinc has been commonly supplemented in the diets of animals to meet their nutritional requirement, promote growth, improve immune function, and reduce the incidence of diarrhea
Corresponding author at: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Beijing, 100193, China. E-mail address: [email protected] (P. Sun). # These authors contributed equally. ⁎
https://doi.org/10.1016/j.livsci.2019.103819 Received 2 February 2019; Received in revised form 11 September 2019; Accepted 4 October 2019 Available online 05 October 2019 1871-1413/ © 2019 Elsevier B.V. All rights reserved.
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(Fairbrother et al., 2005; Pettigrew, 2006). Oral administration of zinc oxide (ZnO), especially at pharmacological doses, improves growth performance and effectively reduces the incidence of diarrhea in weaner pigs (Heo et al., 2010). It is reported that ZnO is the only inorganic form of zinc that produces these benefits of the natural forms of zinc (Schell and Kornegay, 1996; Hu et al., 2012; Glover et al., 2013). However, the use of pharmacological doses of zinc has been strictly prohibited because large amount of zinc may result in high quantity of unabsorbed zinc being excreted in feces. The Ministry of Agriculture and Rural Affairs of China requires that the zinc content of the ration of calves is < 180 mg/kg. Unfortunately, the optimal dose of zinc required by neonatal calves has been unclear until now. Most of previous studies focused on the effects of very large amounts of ZnO supplementation in monogastric animals, whereas investigations of the optimal concentrations of supplementation required to meet the needs of, and especially improve antioxidant status and promote health in, neonatal calves have been few in number. Therefore, we hypothesized that low dose of zinc might affect zinc metabolism and antioxidant status of neonatal calves, which might help reduce the incidence of diarrhea and then promote growth. The present study aimed to evaluate the effects of various doses of ZnO on growth performance, the incidence of diarrhea, zinc contents in feces and serum, antioxidant indicators and the serum key enzymes and proteins involved in zinc metabolism in neonatal dairy calves. This may help to elucidate the optimal amount of supplementation with zinc for dairy calves during their early life in calf rearing.
Table 1 Ingredient composition and nutrient levels of starter diet (dry matter basis). Ingredient composition
%
Corn Chinese wildrye Soybean cake Wheat bran Wheat middlings Molasses Ground oyster shell Salt Bentonite Calcium phosphate Extruded soybean Premixa Chemical analysisb Dry matter, % Crude protein, % Ether extract, % Acid detergent fiber, % Neutral detergent fiber, % Ash, %
37.8 20.2 18.2 2.8 7.8 1.6 1.6 0.8 0.8 1.2 6.4 0.8 89.01 20.83 3.57 5.55 15.97 6.35.
a
Premix provided per kilogram of diet: vitamin A, 1000 000 IU; vitamin D, 270 000 IU; vitamin E, 2 900 IU; Cu, 5 000 mg; Fe, 9 000 mg; Mn, 6 000 mg; Se, 67 mg; I, 227 mg; Co, 20 mg; Mg, 9 800 mg. b Analyzed value.
trial, mixed with 200 ml colostrum or milk, and fed directly to the calves. Thereafter, more colostrum or milk was fed to the calves. The whole trial lasted for 14 days. Fresh water and starter diet were available ad libitum throughout the study period.
2. Materials and methods This study was conducted between October and December 2017 at Hongyun Dairy Professional Cooperative (Xinle city, Hebei Province, China). All procedures were performed using protocols approved by the Institute of Animal Science, Chinese Academy of Agricultural Sciences (Beijing, China). All animals used in the current study were raised according to standards established by the Institute of Animal Science, Chinese Academy of Agricultural Sciences. The health condition of the calves was monitored and recorded after birth and throughout the experimental period.
2.2. Sampling and analysis The calves were weighted at the end of the trial to calculate the weight gain. Dry matter intake (DMI) of milk and starter diet were recorded throughout the whole experiment. Fecal score was recorded every day during the trial and scored 1–4, where 1 was normal, 2 was pasty, 3 was semiliquid, and 4 was liquid (Teixeira et al., 2015). The observer was an experienced stockman who was blinded to the treatment group. Diarrhea was defined as the presence of grade 3 or 4 feces for 2 consecutive days. Diarrhea incidence (%) was calculated as the sum of the number of calves with diarrhea in each group × the number of days with diarrhea / 8 / 14 × 100% (Sun et al., 2008). A sample of starter diet was ground in a fodder grinder using a 1 mm screen. DM content was measured by drying at 105 °C for 4 h to constant-mass (AOAC, 2005; method 930.15), and crude protein (CP) (AOAC, 2000; method 976.05) and ether extract (EE) (AOAC, 2003; method 4.5.05) were quantified using the standard procedures of the Association of Official Analytical Chemists. The neutral detergent fiber (NDF) and the acid detergent fiber (ADF) contents were determined using the procedures of Van Soest et al. (1991). Fresh fecal samples (approximately 3.0 g) were obtained by aseptic collection from the rectum at the end of the trial for subsequent assay of zinc concentration. Blood samples were collected from a jugular vein of all the calves using evacuated tubes (BD Biosciences, San Jose, CA, USA) at the end of the experiment, before the morning feed. Serum was obtained by centrifugation at 3000 × g for 15 min at 4 °C, and then stored at −20 °C for subsequent assay. The concentrations of micronutrients present in the feces and serum were determined as described in the Chinese National Standards (GB 5009.268, China, 2016) with some modifications (Sun et al., 2017). Briefly, samples of fresh feces (0.50 g) or serum (1.00 g) were digested with a mixture of nitric acid and perchloric acids (v/v, 10:1, 10 ml) in an electrically heated digestion block, and then deionized water was added to 50 mL. Thereafter, inductively coupled plasma mass
2.1. Calves, diets and experimental design Forty newborn Holstein dairy calves (10 male and 30 female) with an initial body mass of 40.6 ± 6.7 kg at birth, were assigned to one of five groups with eight calves (two male and six female) in each group. All calves were removed from the dam within 10 min of birth, and were housed in individual pens (1.8 × 1.4 × 1.2 m) bedded with straw and enclosed with iron fences to avoid cross-contamination among calves on the same farm. Their navels were dipped in iodine, and they were fed with 4 L colostrum from a bottle within 1 h of birth, with any residue being administered by esophageal tube. Subsequently, the calves were fed with 2 L milk twice a day from a bottle at 08:30 and 16:00 on days 2 and 3. Thereafter, 8 L milk was fed to the calves from day 4 to 14. A starter diet without added zinc (Beijing Sanyuan Seed Technology Co., Ltd., Beijing, China) was formulated to meet nutrient requirements, according to the National Research Council (2001), and given to the calves at the same time as the milk fed. The composition and nutrient levels of the starter are shown in Table 1. Before the initiation of the trial, the zinc concentrations of the water, milk, and starter were determined. No zinc was detectable in the water, and all calves received the same milk and starter diet, containing basal zinc concentrations of 4.01 mg/kg and 22.97 mg/kg, respectively. All calves were fed diets supplemented with ZnO at 0, 25.79, 51.58, 103.16, or 154.74 mg/d, yielding a zinc content of 0 (Control), 20 (Zn20), 40 (Zn-40), 80 (Zn-80), 120 (Zn-120) mg/d, respectively, during the experimental period. The appropriate amount of ZnO was weighed and separately packed for each calf before the commencement of the 2
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spectrometry/vapor generation (ICP-MS/MS, Agilent 8800, Agilent Co., CA, USA) was used to measure the concentration of microelements. The results are presented as the mean of two replicates that were analyzed in duplicate. Serum concentrations of alkaline phosphatase (ALP) and metallothionein (MT) were determined using ELISA kits (Wuhan ColorfulGene biological technology Co., Ltd., Wuhan, China) according to the manufacturer's instructions. Markers of antioxidant capacity were also assayed in the serum (glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA)) using commercial assay kits (Nanjing Jian Cheng Bioengineering Institute, Nanjing, China), following the instructions of the manufacturer.
content was greater in the Zn-120 group than in the other four groups (P < 0.05). There was no effect of the dose of zinc supplementation on calcium, copper, iron, magnesium, and phosphorus serum concentrations in newborn calves. However, the serum concentration of zinc increased first and then decreased, which presented a quadratic response as the supplementation amount of zinc increased, peaking at 80 mg zinc/d (P < 0.05). 3.3. Serum concentrations of zinc-dependent enzymes, zinc-binding proteins, and antioxidant markers The concentrations of serum ALP, MT, GSH-Px and MDA as well as the SOD activity are presented in Table 4. An increase in dietary ZnO quadratically increased ALP concentration and peaked at 80 mg zinc/d (P < 0.05). Simultaneously, supplementation of ZnO linearly enhanced the concentration of MT (P < 0.01). As shown in Table 4, no differences were observed in GSH-Px concentrations among the treatment groups. Notably, serum SOD activity increased linearly (P < 0.01), but MDA content declined linearly (P < 0.05) as ZnO supplementation dose enhanced.
2.3. Statistical analysis A chi-square contingency test was used to test the effect of zinc supplementation on the prevalence of diarrhea among the treatments using the GLIMMIX procedure of SAS software (version 9.3, SAS Institute Inc., Cary, NC, USA). All other data were analyzed using GLIMMIX procedure of SAS (version 9.3, SAS Institute Inc., Cary, NC, USA). The sex of the calf did not contribute significantly and was therefore not included in the model. Contrasts were constructed to examine the effects of ZnO supplementation levels, with orthogonal polynomials accounting for unequal spacing of zinc supplementation levels. Data are expressed as the least square mean and standard error of the mean. Statistical significance was declared at P < 0.05, and trends were accepted when 0.05 ≤ P < 0.10.
4. Discussion Zinc homoeostasis is maintained primarily by intestinal absorption and fecal or obligate urinary excretion (NRC, 2001). Dietary zinc supply is required because zinc is an essential trace element that cannot be synthesized or stored in the body (Bonaventura et al., 2015). Previous studies demonstrated the beneficial effects of zinc on calf growth (Graham et al., 2010; Glover et al., 2013). Data collected in the current trial showed that a daily intake of < 120 mg did not influence the ADG, ADFI, or feed efficiency of neonatal dairy calves. This result supports previous investigations demonstrating no effect on growth performance of heifers fed diets supplemented with low dose of organic or inorganic zinc (Salyer et al., 2004). Spears et al. (1991) and Galyean et al. (1995) also reported no differences in performance of calves or growing steers when supplemented with organic or inorganic zinc. Considering that the first 2–3 weeks after birth is when the prevalence of diarrhea peaks in calves, we aimed to screen the optimal dose of dietary ZnO supplementation during the first 2 weeks of life to investigate its preventive effect on diarrhea. Unexpectedly, no difference was observed in the incidence of diarrhea after a relatively low amount of zinc supplementation, which was different to those reports in children (Patel et al., 2010). The reason might be that zinc could alleviate diarrhea in children commonly under the deficiency of zinc, which might contribute to the divergence of the results on calves. In the present study, the control calves received zinc from milk and starter, which might approximate their marginal requirements. In the present study, except for diarrhea, no other apparent symptoms of zinc deficiency were observed in the calves from the control group. Although pharmacological doses of ZnO can be similarly effective to
3. Results 3.1. Performance and diarrhea of the calves The effects of ZnO supplementation on the growth performance and incidence of diarrhea are presented in Table 2. In general, supplementation with differing amounts of ZnO did not influence the growth performance or the incidence of diarrhea of the calves (P > 0.05). No difference was observed in their final body weight, average daily gain (ADG), average daily feed intake (ADFI), or feed efficiency. Although not significantly, zinc supplementation numerically reduced the incidence of diarrhea compared with the control group. When zinc supplementation increased to 80 and 120 mg/d, incidence of diarrhea reduced to 22.32% and 21.43%. 3.2. Microelement concentrations in the feces and serum The effects of various amounts of zinc supplementation on fecal and serum micronutrient concentrations are shown in Table 3. The concentrations of fecal zinc and iron linearly increased as the supplementation amount of zinc increased (P < 0.01). Compared with the control group, the fecal zinc content was greater when calves were supplemented with zinc at the dose of 40 and 120 mg/d, and the iron
Table 2 The growth performance and incidence of diarrhea in Holstein dairy calves fed differing amounts of ZnOa. Item
Zinc supplementation amount (mg/d) 0 20 40
80
120
Initial body weight (kg) Final body weight (kg) Average daily gain (g/d) Liquid intake (g DM/d) Starter intake (g DM/d) Total intake (g DM/d) Feed efficiency (g DMI/g gain) Incidence of diarrhea (%)
40.6 46.5 425 839 55.7 895 2.32 33.93
40.5 47.2 476 863 55.1 918 2.24 22.32
40.5 47.1 472 824 51.3 875 2.38 21.43
a
40.7 47.0 452 843 48.1 891 2.24 27.68
40.5 46.0 388 847 55.6 902 2.99 24.11
SEM
P value Linear
Quadratic
3.94 3.62 72.8 17.6 3.99 18.9 0.387 –
0.98 0.89 0.55 0.73 0.86 0.72 0.93 0.16
1.00 0.96 0.86 0.20 0.77 0.21 0.54 0.52
Each value is the mean of data from eight calves per treatment. bSignificantly different by chi-square contingency test. 3
Livestock Science 230 (2019) 103819
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Table 3 The concentrations of micronutrients in feces and serum of Holstein dairy calves fed differing amounts of ZnOa. Item
Zinc supplementation amount (mg/d) 0 20
Content of micronutrient in feces (mg/kg) Zinc 93.76 142.76 Iron 258.23 255.46 Copper 7.26 6.27 Content of micronutrient in serum (mg/kg) Calcium 180.05 180.76 Copper 1.49 1.26 Iron 3.54 3.49 Magnesium 22.32 24.32 Phosphorus 156.43 154.39 Zinc 0.94 1.13 a
SEM
P value Linear
Quadratic
277.13 594.52 9.44
41.980 75.467 1.632
< 0.01 < 0.01 0.32
0.38 0.19 0.89
186.16 1.34 3.32 23.88 166.64 1.12
9.456 0.128 0.560 1.236 8.450 0.101
0.27 0.82 0.60 0.29 0.25 0.10
0.62 0.67 0.17 0.47 0.46 0.02
40
80
120
240.51 349.98 9.82
207.55 306.28 7.65
175.02 1.32 3.50 19.66 149.73 1.16
200.63 1.43 5.01 24.25 160.52 1.40
Each value is the mean of data from eight calves per treatment.
Table 4 Zinc-dependent enzymes, zinc-binding proteins, and antioxidant indicators in the serum of Holstein dairy calves fed differing amounts of ZnOa. Item
Zinc supplementation amount (mg/d) 0 20 40
80
120
Alkaline phosphatase (pg/mL) Metallothionein (pg/mL) Glutathione peroxidase (nmol/mL) Superoxide dismutase (U/mL) Malondialdehyde (nmol/mL)
1509.54 833.59 162.47 76.25 7.12
1805.48 979.36 180.46 82.55 5.53
1729.38 996.65 189.16 87.90 5.12
a
1691.03 887.45 168.36 79.40 5.85
1772.63 925.52 175.26 80.44 5.73
SEM
P value Linear
Quadratic
79.319 46.414 14.634 4.225 0.578
0.07 < 0.01 0.17 < 0.01 0.03
0.04 0.42 0.89 0.88 0.35
Each value is the mean of data from eight calves per treatment.
Another important zinc-dependent enzyme is SOD, which is an extremely effective antioxidant metalloenzyme. It is well known that SOD catalyzes the dismutation of superoxide anion radicals to produce hydrogen peroxide, which is considered to be an important marker of the antioxidant status of animals (Prasad, 2008; Gong et al., 2014). Cunnigham-Rundles et al. (1990) found that SOD activity reached a peak at a dietary zinc content of 80–120 mg/kg. In addition, zinc may induce the synthesis of MT, a group of metal-binding proteins with high affinity for zinc, and which are excellent scavengers of ∙OH, indeed far superior to SOD and GSH-Px (Kagi and Schaffer, 1998; Mocchegiani et al., 2011). Conversely, zinc also plays a significant role in the reduction of MDA concentrations (Karamouz et al., 2010), which is one of the end-products of lipid peroxidation and is also a useful marker of oxidative stress (Gaweł et al., 2004). All of these publications suggest that zinc is an effective antioxidant, and many studies conducted in cell culture and animal models have corroborated this (Karamouz et al., 2010). In agreement with these publications, the current study showed that the concentration of MT and the activity of SOD were linearly increased and the serum MDA content was linearly declined as the dose of dietary ZnO increased, especially for amount of > 80 mg/d. No differences were observed in GSH-Px activity, implying that zinc did not affect its activity under the conditions of the present study. The improved antioxidant status of dairy calves fed 80 mg zinc/d might contribute to the reduction in the prevalence of diarrhea, but the mechanism of this effect requires further investigation.
antibiotics, the use of excessive amounts of zinc waste resources and may interact with other metal ions (NRC, 2001; Glover et al., 2013; Deng et al., 2017). In this study, supplementation with zinc quadratically increased serum zinc concentration peaking at 80 mg/d, but did not result in changes in the iron or copper content in the serum. Similarly, Jia et al. (2009) also did not observed difference of zinc supplementation on serum concentrations of copper and iron in Cashmere goats. Furthermore, supplementation of increasing doses of zinc had no effect on serum calcium, phosphorus and magnesium concentrations among different groups. Our results were supported by Garg et al. (2008) who found that zinc supplementation did not influence serum calcium, inorganic phosphorus and manganese concentrations in the lambs. The optimal zinc content of a feed would maximize the health and nutritional benefit and keep balance with other micronutrients. Under the conditions of the present study, dietary zinc supplementation did not interferes with the absorption of copper, although zinc and copper are antagonistic to one another (NRC, 2001). In the present study, fecal zinc and iron contents were linearly increased as the supplementation doses of ZnO in the diet enhanced, which furtherly proved that iron and zinc might share a common absorption mechanism (NRC, 2001). Compared with other ZnO supplementation doses, calves fed 80 mg zinc/d had a relatively low fecal zinc content despite a greater serum zinc content, which might indicate greater bioavailability. It has been proposed that the serum concentrations of zinc-dependent enzymes (including ALP and SOD) and zinc-binding proteins (such as MT and insulin) are good indicators of zinc nutritional status in addition to serum and tissue zinc contents (Vallee and Falchuk, 1993; Yin et al., 2009). As one of the zinc-dependent enzymes, ALP is a sensitive indicator that reflects zinc concentrations in the body. Samman et al. (1996) reported that ALP activity in erythrocytes decreased as a result of low dietary zinc content. In the present study, the concentration of ALP increased quadratically with the increasing amount of zinc supplementation, peaking at 80 mg/d, which was consistent with the effects of supplementation on serum zinc content. Similar results were also obtained by Ruz et al. (1992), who suggested that ALP might be a potential index of zinc status in humans.
5. Conclusions The present study showed that low amounts of ZnO supplementation did not influence growth performance or the incidence of diarrhea. However, the serum and fecal zinc concentrations increased as the ZnO supplementation amount increased. Calves fed 80 mg zinc/d had a relatively lesser fecal zinc content and a greater serum zinc content, which might indicate relatively greater bioavailability at this amount of zinc supplementation. Dietary supplementation with ZnO also increased ALP, SOD and MT concentrations and decreased MDA content 4
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in the serum, suggesting that zinc improved antioxidant status in neonatal dairy calves, which might in turn contribute to the numerical lesser diarrhea incidence. This study suggested that a zinc supplementation amount of 80 mg/d in the form of ZnO has high bioavailability, which may be recommended for newborn calves to promote zinc metabolism.
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Declaration of Competing Interest The authors have no conflict of interest. Acknowledgments This study was financially supported by the National Key Research and Development Program of China (2016YFD0500507, 2018YFD0500703), the Agriculture Science and Technology Innovation Program(ASTIP-IAS07), and the National Program for Support of Topnotch Young Professionals. References Bonaventura, P., Benedetti, G., Albarède, F., Miossec, P., 2015. Zinc and its role in immunity and inflammation. Autoimmun. Rev. 14 (4), 277–285. Cunnigham-Rundles, S., Bockman, R.S., Lin, A., Giardina, P.V., Hilgartner, M.W., Caldwell-Brown, D., Carter, D.M., 1990. Physiological and pharmacological effects of zinc on immune response. Ann. N.Y Acad. Sci. 587, 113–122. Deng, B., Zhou, X., Wu, J., Long, C., Yao, Y., Peng, H., Wan, D., Wu, X., 2017. Effects of dietary supplementation with tribasic zinc sulfate or zinc sulfate on growth performance, zinc content and expression of zinc transporters in young pigs. Anim. Sci. J. 88, 1556–1560. Dresler, S., Illek, J., Zeman, L., 2016. Effects of organic zinc supplementation in weaned calves. Acta Vet. Brno 85, 49–54. Fairbrother, J.M., Nadeau, É., Gyles, C.L., 2005. Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesisi, and prevention strategies. Anim. Health Res. Rev 6, 17–39. Galyean, M.L., Malcolm-Callis, K.J., Gunter, S.A., Berrie, R.A., 1995. Effect of zinc source and level and added copper lysine in the receiving diet on performance by growing an dfinishing steers. Prof. Anim. Sci. 11, 139–148. Garg, A.K., Mudgal, Vishal, Dass, R.S., 2008. Effect of organic zinc supplementasion on growth, nutrient utilization and mineral profile in lambs. Anim. Feed Sci. Technol. 144, 82–96. Gaweł, S., Wardas, M., Niedworok, E., Wardas, P., 2004. Malondialdehyde (MDA) as a lipid peroxidation marker. Wiad. Lek. 57, 453–455. Graham, T.W., Breher, J.E., Farver, T.B., Cullor, J.S., Kehrli, M.E., Oberbauer, A.M., 2010. Biological markers of neonatal calf performance: the relationship of insulin-like growth factor-I, zinc, and copper to poor neonatal growth. J. Anim. Sci. 88, 2585–2593. Glover, A.D., Puschner, B., Rossow, H.A., Lehenbauer, T.W., Champagne, J.D., Blanchard, P.C., Aly, S.S., 2013. A double-blind block randomized clinical trial on the effect of zinc as a treatment for diarrhea in neonatal Holstein calves under natural challenge conditions. Prev. Vet. Med. 112 (3–4), 338–347. Gong, J., Ni, L., Wang, D., Shi, B., Yan, S., 2014. Effect of dietary organic selenium on milk selenium concentration and antioxidant and immune status in midlactation dairy cows. Livest. Sci. 170, 84–90. Heo, J.M., Kim, J.C., Hansen, C.F., Mullan, B.P., Hampson, D.J., Maribo, H., Kjeldsen, N., Pluske, J.R., 2010. Effects of dietary protein level and zinc oxide supplementation on the incidence of post-weaning diarhoea in weaner pigs challenged with an enterotoxigenic strain of Escherichia coli. Livest. Sci. 133, 210–213. Hu, C.H., Gu, L.Y., Luan, Z.S., Song, J., Zhu, K., 2012. Effects of montmorillonite-zinc oxide hybrid on performance, diarrhea, intestinal permeability and morphology of weaning pigs. Anim. Feed Sci. Technol. 177, 108–115. Hu, C.H., Xiao, K., Song, J., Luan, Z.S., 2013. Effects of zinc oxide supported on zeolite on growth performance, intestinal microflora and permeability, and cytokines expression of weaned pigs. Anim. Feed Sci Technol. 181, 65–71. Jia, W., Zhu, X., Zhang, W., Cheng, J., Guo, C., Jia, Z., 2009. Effects of source of supplemental zinc on performance, nutrient digestibility and plasma mineal profile in cashmere goats. Asian-Aust. J. Anim. Sci. 22, 1648–1653. Kagi, J.H., Schaffer, A., 1998. Biochemistry of metallothionein. Biochemistry 127, 8509–8515. Karamouz, H., Shahriyar, H.A., Gorbani, A., Maheri-Sis, N., Ghaleh-kandi, J.G., 2010. Effect of zinc oxide supplementation on some serum biochemical values in male
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Effect of differing amounts of zinc oxide supplementation on the antioxidant status and zinc metabolism in newborn dairy calves Jingya Wei#, Fengtao Ma#, Liyuan Hao, Qiang Shan, Peng Sun
T
⁎
State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
ARTICLE INFO
ABSTRACT
Keywords: Zinc oxide Zinc metabolism Antioxidant status Dairy calf
This study aimed to evaluate the effects of various amounts of zinc oxide (ZnO) supplementation on growth performance, incidence of diarrhea, micronutrient contents in feces and serum, antioxidant indicators and the concentrations of key enzymes and proteins involved in zinc metabolism in dairy calves. Forty newborn Holstein dairy calves were assigned to one of five groups according to a randomized complete block design: a control group (without zinc supplementation), or groups that received ZnO supplementation at 25.79, 51.58, 103.16, and 154.74 mg/d, yielding an amount of zinc consumption of 20 (Zn-20), 40 (Zn-40), 80 (Zn-80), and 120 (Zn120) mg/d, respectively, during the experimental period. The experiment lasted for 14 days, and blood and fecal samples were collected at the end of the trial. Supplementation with differing amounts of ZnO did not influence the growth performance or incidence of diarrhea of the calves (P > 0.05). However, the concentrations of fecal zinc and iron linearly increased with the amount of ZnO supplementation (P < 0.01). Compared with the control group, fecal zinc content was greater when calves were supplemented with zinc at concentrations of 40 and 120 mg/d, but the iron content was greater in the Zn-120 group than in the other four groups (P < 0.05). The amount of zinc supplementation did not affect the serum concentrations of micronutrients, including calcium, copper, iron, magnesium and phosphorus, in the newborn calves (P > 0.05). While the serum zinc concentration increased quadratically as the amount of supplementation increased, peaking with 80 mg zinc/d supplementation (P < 0.05). Increasing doses of ZnO supplementation linearly elevated the concentrations of metallothionein and superoxide dismutase (P < 0.01), and quadratically enhanced that of alkaline phosphatase (P < 0.05), but linearly reduced that of malondialdehyde in serum (P < 0.05). This study demonstrates that dietary ZnO supplementation to yield 80 mg zinc/d improves zinc metabolism and antioxidant status of newborn calves during their early life.
1. Introduction Diarrhea is one of the most important health problems in young calves, especially in neonatal calves (Virtala et al., 1996; Mayer et al., 2012), which is considered one of the principal causes of financial loss for dairy farms (Pourliotis et al., 2012). Zinc, a trace element, has many biological functions, including the maintenance of epithelia barrier integrity, cell division, and a sufficient immune response (Liberato et al., 2015; Schulte et al., 2016). Importantly, it has also been shown that zinc is an effective anti-inflammatory and anti-diarrheal agent (Oteiza and Mackenzie, 2005; Hu et al., 2013; Bonaventura et al., 2015). As the most effective method of administering zinc, oral Zn supplementation has been used globally in the prevention and treatment of diarrhea in infants and children, and millions of lives have been
saved in recent decades as a result (Prasad, 2014; Liberato et al., 2015). Simultaneously, the role of zinc in modulating oxidative stress has also been recognized (Prasad et al., 2004). Previous studies show that zinc supplementation can correct the increased plasma oxidative stress markers (Prasad, 2008). Dresler et al. (2016) reported that zinc methionine (Zn-Met) increased serum SOD activity in weaned female calves. Saleh et al. (2018) found that Zn-Met enhanced plasma glutathione peroxidase (GSH-Px) concentration compared with control in broilers exposed to high ambient temperature. Li et al. (2019) demonstrated that Zn-Met at 60 mg/kg elevated GSH-Px activity in serum and the SOD and GSH-Px activity in liver of laying hens. For decades, zinc has been commonly supplemented in the diets of animals to meet their nutritional requirement, promote growth, improve immune function, and reduce the incidence of diarrhea
Corresponding author at: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Beijing, 100193, China. E-mail address: [email protected] (P. Sun). # These authors contributed equally. ⁎
https://doi.org/10.1016/j.livsci.2019.103819 Received 2 February 2019; Received in revised form 11 September 2019; Accepted 4 October 2019 Available online 05 October 2019 1871-1413/ © 2019 Elsevier B.V. All rights reserved.
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(Fairbrother et al., 2005; Pettigrew, 2006). Oral administration of zinc oxide (ZnO), especially at pharmacological doses, improves growth performance and effectively reduces the incidence of diarrhea in weaner pigs (Heo et al., 2010). It is reported that ZnO is the only inorganic form of zinc that produces these benefits of the natural forms of zinc (Schell and Kornegay, 1996; Hu et al., 2012; Glover et al., 2013). However, the use of pharmacological doses of zinc has been strictly prohibited because large amount of zinc may result in high quantity of unabsorbed zinc being excreted in feces. The Ministry of Agriculture and Rural Affairs of China requires that the zinc content of the ration of calves is < 180 mg/kg. Unfortunately, the optimal dose of zinc required by neonatal calves has been unclear until now. Most of previous studies focused on the effects of very large amounts of ZnO supplementation in monogastric animals, whereas investigations of the optimal concentrations of supplementation required to meet the needs of, and especially improve antioxidant status and promote health in, neonatal calves have been few in number. Therefore, we hypothesized that low dose of zinc might affect zinc metabolism and antioxidant status of neonatal calves, which might help reduce the incidence of diarrhea and then promote growth. The present study aimed to evaluate the effects of various doses of ZnO on growth performance, the incidence of diarrhea, zinc contents in feces and serum, antioxidant indicators and the serum key enzymes and proteins involved in zinc metabolism in neonatal dairy calves. This may help to elucidate the optimal amount of supplementation with zinc for dairy calves during their early life in calf rearing.
Table 1 Ingredient composition and nutrient levels of starter diet (dry matter basis). Ingredient composition
%
Corn Chinese wildrye Soybean cake Wheat bran Wheat middlings Molasses Ground oyster shell Salt Bentonite Calcium phosphate Extruded soybean Premixa Chemical analysisb Dry matter, % Crude protein, % Ether extract, % Acid detergent fiber, % Neutral detergent fiber, % Ash, %
37.8 20.2 18.2 2.8 7.8 1.6 1.6 0.8 0.8 1.2 6.4 0.8 89.01 20.83 3.57 5.55 15.97 6.35.
a
Premix provided per kilogram of diet: vitamin A, 1000 000 IU; vitamin D, 270 000 IU; vitamin E, 2 900 IU; Cu, 5 000 mg; Fe, 9 000 mg; Mn, 6 000 mg; Se, 67 mg; I, 227 mg; Co, 20 mg; Mg, 9 800 mg. b Analyzed value.
trial, mixed with 200 ml colostrum or milk, and fed directly to the calves. Thereafter, more colostrum or milk was fed to the calves. The whole trial lasted for 14 days. Fresh water and starter diet were available ad libitum throughout the study period.
2. Materials and methods This study was conducted between October and December 2017 at Hongyun Dairy Professional Cooperative (Xinle city, Hebei Province, China). All procedures were performed using protocols approved by the Institute of Animal Science, Chinese Academy of Agricultural Sciences (Beijing, China). All animals used in the current study were raised according to standards established by the Institute of Animal Science, Chinese Academy of Agricultural Sciences. The health condition of the calves was monitored and recorded after birth and throughout the experimental period.
2.2. Sampling and analysis The calves were weighted at the end of the trial to calculate the weight gain. Dry matter intake (DMI) of milk and starter diet were recorded throughout the whole experiment. Fecal score was recorded every day during the trial and scored 1–4, where 1 was normal, 2 was pasty, 3 was semiliquid, and 4 was liquid (Teixeira et al., 2015). The observer was an experienced stockman who was blinded to the treatment group. Diarrhea was defined as the presence of grade 3 or 4 feces for 2 consecutive days. Diarrhea incidence (%) was calculated as the sum of the number of calves with diarrhea in each group × the number of days with diarrhea / 8 / 14 × 100% (Sun et al., 2008). A sample of starter diet was ground in a fodder grinder using a 1 mm screen. DM content was measured by drying at 105 °C for 4 h to constant-mass (AOAC, 2005; method 930.15), and crude protein (CP) (AOAC, 2000; method 976.05) and ether extract (EE) (AOAC, 2003; method 4.5.05) were quantified using the standard procedures of the Association of Official Analytical Chemists. The neutral detergent fiber (NDF) and the acid detergent fiber (ADF) contents were determined using the procedures of Van Soest et al. (1991). Fresh fecal samples (approximately 3.0 g) were obtained by aseptic collection from the rectum at the end of the trial for subsequent assay of zinc concentration. Blood samples were collected from a jugular vein of all the calves using evacuated tubes (BD Biosciences, San Jose, CA, USA) at the end of the experiment, before the morning feed. Serum was obtained by centrifugation at 3000 × g for 15 min at 4 °C, and then stored at −20 °C for subsequent assay. The concentrations of micronutrients present in the feces and serum were determined as described in the Chinese National Standards (GB 5009.268, China, 2016) with some modifications (Sun et al., 2017). Briefly, samples of fresh feces (0.50 g) or serum (1.00 g) were digested with a mixture of nitric acid and perchloric acids (v/v, 10:1, 10 ml) in an electrically heated digestion block, and then deionized water was added to 50 mL. Thereafter, inductively coupled plasma mass
2.1. Calves, diets and experimental design Forty newborn Holstein dairy calves (10 male and 30 female) with an initial body mass of 40.6 ± 6.7 kg at birth, were assigned to one of five groups with eight calves (two male and six female) in each group. All calves were removed from the dam within 10 min of birth, and were housed in individual pens (1.8 × 1.4 × 1.2 m) bedded with straw and enclosed with iron fences to avoid cross-contamination among calves on the same farm. Their navels were dipped in iodine, and they were fed with 4 L colostrum from a bottle within 1 h of birth, with any residue being administered by esophageal tube. Subsequently, the calves were fed with 2 L milk twice a day from a bottle at 08:30 and 16:00 on days 2 and 3. Thereafter, 8 L milk was fed to the calves from day 4 to 14. A starter diet without added zinc (Beijing Sanyuan Seed Technology Co., Ltd., Beijing, China) was formulated to meet nutrient requirements, according to the National Research Council (2001), and given to the calves at the same time as the milk fed. The composition and nutrient levels of the starter are shown in Table 1. Before the initiation of the trial, the zinc concentrations of the water, milk, and starter were determined. No zinc was detectable in the water, and all calves received the same milk and starter diet, containing basal zinc concentrations of 4.01 mg/kg and 22.97 mg/kg, respectively. All calves were fed diets supplemented with ZnO at 0, 25.79, 51.58, 103.16, or 154.74 mg/d, yielding a zinc content of 0 (Control), 20 (Zn20), 40 (Zn-40), 80 (Zn-80), 120 (Zn-120) mg/d, respectively, during the experimental period. The appropriate amount of ZnO was weighed and separately packed for each calf before the commencement of the 2
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spectrometry/vapor generation (ICP-MS/MS, Agilent 8800, Agilent Co., CA, USA) was used to measure the concentration of microelements. The results are presented as the mean of two replicates that were analyzed in duplicate. Serum concentrations of alkaline phosphatase (ALP) and metallothionein (MT) were determined using ELISA kits (Wuhan ColorfulGene biological technology Co., Ltd., Wuhan, China) according to the manufacturer's instructions. Markers of antioxidant capacity were also assayed in the serum (glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA)) using commercial assay kits (Nanjing Jian Cheng Bioengineering Institute, Nanjing, China), following the instructions of the manufacturer.
content was greater in the Zn-120 group than in the other four groups (P < 0.05). There was no effect of the dose of zinc supplementation on calcium, copper, iron, magnesium, and phosphorus serum concentrations in newborn calves. However, the serum concentration of zinc increased first and then decreased, which presented a quadratic response as the supplementation amount of zinc increased, peaking at 80 mg zinc/d (P < 0.05). 3.3. Serum concentrations of zinc-dependent enzymes, zinc-binding proteins, and antioxidant markers The concentrations of serum ALP, MT, GSH-Px and MDA as well as the SOD activity are presented in Table 4. An increase in dietary ZnO quadratically increased ALP concentration and peaked at 80 mg zinc/d (P < 0.05). Simultaneously, supplementation of ZnO linearly enhanced the concentration of MT (P < 0.01). As shown in Table 4, no differences were observed in GSH-Px concentrations among the treatment groups. Notably, serum SOD activity increased linearly (P < 0.01), but MDA content declined linearly (P < 0.05) as ZnO supplementation dose enhanced.
2.3. Statistical analysis A chi-square contingency test was used to test the effect of zinc supplementation on the prevalence of diarrhea among the treatments using the GLIMMIX procedure of SAS software (version 9.3, SAS Institute Inc., Cary, NC, USA). All other data were analyzed using GLIMMIX procedure of SAS (version 9.3, SAS Institute Inc., Cary, NC, USA). The sex of the calf did not contribute significantly and was therefore not included in the model. Contrasts were constructed to examine the effects of ZnO supplementation levels, with orthogonal polynomials accounting for unequal spacing of zinc supplementation levels. Data are expressed as the least square mean and standard error of the mean. Statistical significance was declared at P < 0.05, and trends were accepted when 0.05 ≤ P < 0.10.
4. Discussion Zinc homoeostasis is maintained primarily by intestinal absorption and fecal or obligate urinary excretion (NRC, 2001). Dietary zinc supply is required because zinc is an essential trace element that cannot be synthesized or stored in the body (Bonaventura et al., 2015). Previous studies demonstrated the beneficial effects of zinc on calf growth (Graham et al., 2010; Glover et al., 2013). Data collected in the current trial showed that a daily intake of < 120 mg did not influence the ADG, ADFI, or feed efficiency of neonatal dairy calves. This result supports previous investigations demonstrating no effect on growth performance of heifers fed diets supplemented with low dose of organic or inorganic zinc (Salyer et al., 2004). Spears et al. (1991) and Galyean et al. (1995) also reported no differences in performance of calves or growing steers when supplemented with organic or inorganic zinc. Considering that the first 2–3 weeks after birth is when the prevalence of diarrhea peaks in calves, we aimed to screen the optimal dose of dietary ZnO supplementation during the first 2 weeks of life to investigate its preventive effect on diarrhea. Unexpectedly, no difference was observed in the incidence of diarrhea after a relatively low amount of zinc supplementation, which was different to those reports in children (Patel et al., 2010). The reason might be that zinc could alleviate diarrhea in children commonly under the deficiency of zinc, which might contribute to the divergence of the results on calves. In the present study, the control calves received zinc from milk and starter, which might approximate their marginal requirements. In the present study, except for diarrhea, no other apparent symptoms of zinc deficiency were observed in the calves from the control group. Although pharmacological doses of ZnO can be similarly effective to
3. Results 3.1. Performance and diarrhea of the calves The effects of ZnO supplementation on the growth performance and incidence of diarrhea are presented in Table 2. In general, supplementation with differing amounts of ZnO did not influence the growth performance or the incidence of diarrhea of the calves (P > 0.05). No difference was observed in their final body weight, average daily gain (ADG), average daily feed intake (ADFI), or feed efficiency. Although not significantly, zinc supplementation numerically reduced the incidence of diarrhea compared with the control group. When zinc supplementation increased to 80 and 120 mg/d, incidence of diarrhea reduced to 22.32% and 21.43%. 3.2. Microelement concentrations in the feces and serum The effects of various amounts of zinc supplementation on fecal and serum micronutrient concentrations are shown in Table 3. The concentrations of fecal zinc and iron linearly increased as the supplementation amount of zinc increased (P < 0.01). Compared with the control group, the fecal zinc content was greater when calves were supplemented with zinc at the dose of 40 and 120 mg/d, and the iron
Table 2 The growth performance and incidence of diarrhea in Holstein dairy calves fed differing amounts of ZnOa. Item
Zinc supplementation amount (mg/d) 0 20 40
80
120
Initial body weight (kg) Final body weight (kg) Average daily gain (g/d) Liquid intake (g DM/d) Starter intake (g DM/d) Total intake (g DM/d) Feed efficiency (g DMI/g gain) Incidence of diarrhea (%)
40.6 46.5 425 839 55.7 895 2.32 33.93
40.5 47.2 476 863 55.1 918 2.24 22.32
40.5 47.1 472 824 51.3 875 2.38 21.43
a
40.7 47.0 452 843 48.1 891 2.24 27.68
40.5 46.0 388 847 55.6 902 2.99 24.11
SEM
P value Linear
Quadratic
3.94 3.62 72.8 17.6 3.99 18.9 0.387 –
0.98 0.89 0.55 0.73 0.86 0.72 0.93 0.16
1.00 0.96 0.86 0.20 0.77 0.21 0.54 0.52
Each value is the mean of data from eight calves per treatment. bSignificantly different by chi-square contingency test. 3
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Table 3 The concentrations of micronutrients in feces and serum of Holstein dairy calves fed differing amounts of ZnOa. Item
Zinc supplementation amount (mg/d) 0 20
Content of micronutrient in feces (mg/kg) Zinc 93.76 142.76 Iron 258.23 255.46 Copper 7.26 6.27 Content of micronutrient in serum (mg/kg) Calcium 180.05 180.76 Copper 1.49 1.26 Iron 3.54 3.49 Magnesium 22.32 24.32 Phosphorus 156.43 154.39 Zinc 0.94 1.13 a
SEM
P value Linear
Quadratic
277.13 594.52 9.44
41.980 75.467 1.632
< 0.01 < 0.01 0.32
0.38 0.19 0.89
186.16 1.34 3.32 23.88 166.64 1.12
9.456 0.128 0.560 1.236 8.450 0.101
0.27 0.82 0.60 0.29 0.25 0.10
0.62 0.67 0.17 0.47 0.46 0.02
40
80
120
240.51 349.98 9.82
207.55 306.28 7.65
175.02 1.32 3.50 19.66 149.73 1.16
200.63 1.43 5.01 24.25 160.52 1.40
Each value is the mean of data from eight calves per treatment.
Table 4 Zinc-dependent enzymes, zinc-binding proteins, and antioxidant indicators in the serum of Holstein dairy calves fed differing amounts of ZnOa. Item
Zinc supplementation amount (mg/d) 0 20 40
80
120
Alkaline phosphatase (pg/mL) Metallothionein (pg/mL) Glutathione peroxidase (nmol/mL) Superoxide dismutase (U/mL) Malondialdehyde (nmol/mL)
1509.54 833.59 162.47 76.25 7.12
1805.48 979.36 180.46 82.55 5.53
1729.38 996.65 189.16 87.90 5.12
a
1691.03 887.45 168.36 79.40 5.85
1772.63 925.52 175.26 80.44 5.73
SEM
P value Linear
Quadratic
79.319 46.414 14.634 4.225 0.578
0.07 < 0.01 0.17 < 0.01 0.03
0.04 0.42 0.89 0.88 0.35
Each value is the mean of data from eight calves per treatment.
Another important zinc-dependent enzyme is SOD, which is an extremely effective antioxidant metalloenzyme. It is well known that SOD catalyzes the dismutation of superoxide anion radicals to produce hydrogen peroxide, which is considered to be an important marker of the antioxidant status of animals (Prasad, 2008; Gong et al., 2014). Cunnigham-Rundles et al. (1990) found that SOD activity reached a peak at a dietary zinc content of 80–120 mg/kg. In addition, zinc may induce the synthesis of MT, a group of metal-binding proteins with high affinity for zinc, and which are excellent scavengers of ∙OH, indeed far superior to SOD and GSH-Px (Kagi and Schaffer, 1998; Mocchegiani et al., 2011). Conversely, zinc also plays a significant role in the reduction of MDA concentrations (Karamouz et al., 2010), which is one of the end-products of lipid peroxidation and is also a useful marker of oxidative stress (Gaweł et al., 2004). All of these publications suggest that zinc is an effective antioxidant, and many studies conducted in cell culture and animal models have corroborated this (Karamouz et al., 2010). In agreement with these publications, the current study showed that the concentration of MT and the activity of SOD were linearly increased and the serum MDA content was linearly declined as the dose of dietary ZnO increased, especially for amount of > 80 mg/d. No differences were observed in GSH-Px activity, implying that zinc did not affect its activity under the conditions of the present study. The improved antioxidant status of dairy calves fed 80 mg zinc/d might contribute to the reduction in the prevalence of diarrhea, but the mechanism of this effect requires further investigation.
antibiotics, the use of excessive amounts of zinc waste resources and may interact with other metal ions (NRC, 2001; Glover et al., 2013; Deng et al., 2017). In this study, supplementation with zinc quadratically increased serum zinc concentration peaking at 80 mg/d, but did not result in changes in the iron or copper content in the serum. Similarly, Jia et al. (2009) also did not observed difference of zinc supplementation on serum concentrations of copper and iron in Cashmere goats. Furthermore, supplementation of increasing doses of zinc had no effect on serum calcium, phosphorus and magnesium concentrations among different groups. Our results were supported by Garg et al. (2008) who found that zinc supplementation did not influence serum calcium, inorganic phosphorus and manganese concentrations in the lambs. The optimal zinc content of a feed would maximize the health and nutritional benefit and keep balance with other micronutrients. Under the conditions of the present study, dietary zinc supplementation did not interferes with the absorption of copper, although zinc and copper are antagonistic to one another (NRC, 2001). In the present study, fecal zinc and iron contents were linearly increased as the supplementation doses of ZnO in the diet enhanced, which furtherly proved that iron and zinc might share a common absorption mechanism (NRC, 2001). Compared with other ZnO supplementation doses, calves fed 80 mg zinc/d had a relatively low fecal zinc content despite a greater serum zinc content, which might indicate greater bioavailability. It has been proposed that the serum concentrations of zinc-dependent enzymes (including ALP and SOD) and zinc-binding proteins (such as MT and insulin) are good indicators of zinc nutritional status in addition to serum and tissue zinc contents (Vallee and Falchuk, 1993; Yin et al., 2009). As one of the zinc-dependent enzymes, ALP is a sensitive indicator that reflects zinc concentrations in the body. Samman et al. (1996) reported that ALP activity in erythrocytes decreased as a result of low dietary zinc content. In the present study, the concentration of ALP increased quadratically with the increasing amount of zinc supplementation, peaking at 80 mg/d, which was consistent with the effects of supplementation on serum zinc content. Similar results were also obtained by Ruz et al. (1992), who suggested that ALP might be a potential index of zinc status in humans.
5. Conclusions The present study showed that low amounts of ZnO supplementation did not influence growth performance or the incidence of diarrhea. However, the serum and fecal zinc concentrations increased as the ZnO supplementation amount increased. Calves fed 80 mg zinc/d had a relatively lesser fecal zinc content and a greater serum zinc content, which might indicate relatively greater bioavailability at this amount of zinc supplementation. Dietary supplementation with ZnO also increased ALP, SOD and MT concentrations and decreased MDA content 4
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in the serum, suggesting that zinc improved antioxidant status in neonatal dairy calves, which might in turn contribute to the numerical lesser diarrhea incidence. This study suggested that a zinc supplementation amount of 80 mg/d in the form of ZnO has high bioavailability, which may be recommended for newborn calves to promote zinc metabolism.
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