Effects of zinc supplementation on growth performance, blood metabolites and lameness in young Holstein bulls

Abstract

One hundred and six young Holstein bulls (initial body weight = 376.7±18.3 kg) were randomly allocated in two treatments in a completely randomised design for 56 days. Dietary treatments consisted of (1) the basal diet (control; n=50); (2) basal diet plus 150 mg of Zn/kg of dry matter as ZnSO₄ (n=56). Animals received a fresh total mixed ration at 09.00, 12.00 and 15.00 hours for ad libitum intake allowing 10% orts. Group dry matter intake was measured daily. On days 0, 28 and 56, body weights were recorded and blood samples were collected. Liver samples were taken on day 56. All claws of young bulls were examined every two weeks to identify claw lesions. Supplemental Zn decreased the average daily gain (ADG; p<0.01), gain:feed (G:F; p<0.001) and apparent DM digestibility (p<0.001); however, dry matter intake was not influenced. Plasma total protein (p<0.01), urea nitrogen (p<0.01) and albumin (p< 0.05) were higher in animals fed ZnSO₄; however, serum cholesterol concentration decreased in Zn-supplemented animals. The prevalence of lameness (LN) was higher in control group than ZnSO₄-supplemented treatment (28% vs. 12.5%; odds ratio or OR = 2.7; p<0.05). In summary, supplemental Zn decreased ADG and G:F, however, it could decrease the prevalence of LN.

Keywords:

• zinc supplementation
• growth performance
• blood metabolites
• lameness
• young bull

Introduction

Zinc is an essential trace mineral that serves some roles in an animal's body such as the controlling of growth by affecting on feed intake, secretion of mitogenic hormones, gene expression of proteins (Huerta et al. 2002). Also, immunity can be affected by Zn availability in tissues, such as plasma (Cousins and Leinart 1988). Zinc may improve hoof scores for texture (Moore et al. 1988) and also may improve hoof quality measurements (Kessler et al. 2003). The consumption of conventional antibiotics such as tetracyclines is very restricted in Iran, and it is more attractive to use mineral supplementations (as organic or inorganic), such as Zn or others as replacer to control animal's diseases.

High dietary levels of Zn can increase the concentration of Zn in tissues (Kincaid et al. 1976). Fortification of feedlot diets with trace minerals is a common practice. High levels of Zn may result in lameness (LN) decrease; but, it is important to evaluate the growth performance when animal producers like to improve their animal health by using trace minerals such as Zn in high levels. Hatfield et al. (2001) indicated that high levels of supplemental Zn may do so without the risk of adversely affecting the animal's Cu status. Wellington et al. (1998) stated that caution must be exercised when supplementing excess Zn without Cu to avoid compromising the animal's Cu status. Also, deficiencies and imbalances of trace elements can affect productivity of ruminants. Therefore, the present study was conducted to determine the effect of Zn supplementation on growth performance, blood metabolites, liver Zn concentration and claw health in young Holstein bulls in which the Cu and Fe levels in the basal diet was less than the NRC (2000) recommendations because of evaluating growth and other parameters.

Materials and methods

Animal care and experimental design

The protocol for this research was approved by the University of Zanjan Institutional Animal Care and Use Committee. The basal diet provided to the young bulls was formulated to meet the requirements for finishing period (NRC 2000; Table 1). However, the copper content in the basal diet was marginal to evaluate its effects on claw health and performance. One hundred and six young Holstein bulls (initial Body Weight = 376.7±18.3 kg) were randomly allocated in two treatments in a completely randomised design for 56 days. Treatment groups received (1) the basal diet with no supplemental Zn (control); (2) the basal diet plus 150 mg of Zn/kg of DM as ZnSO₄. Animals were randomly grouped in six pens (16–19 animals per pen). The chemical composition and nutritive value of diet components are shown in Table 1. The control diet contained 44.8 mg of native Zn per kg of DM. Animals received a fresh total mixed ration at 09.00, 12.00 and 15.00 hours, as times of feeding, for ad libitum intake allowing 10% orts. Also, water was available ad libitum. ZnSO₄, which was mixed in a wheat bran carrier, was fed after morning meal as a top-dress.

Table 1. Composition of basal diet.

	Total mixed ration
Ingredient	Percentage of DM
Corn silage	7.5
Wheat straw	2
Barley (milled)	54
Potato	12
Cottonseed meal	3
Sunflower meal	4.78
Wheat bran	4
Rice bran	6
Beet pulp	2
Molasses	1.5
Zeolite	1.5
Urea	0.28
Calcium carbonate	0.56
Salt	0.28
Vitamin premix^a	0.3
Mineral premix^b	0.3
Chemical composition	Percentage of DM
CP	13.25
NDF	29.25
ADF	12.06
Ether extract	3.04
Ca	0.63
P	0.24
Mg	0.31
S	0.14
	mg/kg DM
Cu	4.7
Zn	44.8
Mn	59
Fe	30.2
Mo	1.2
^aContained per kilogram of premix: 500,000 IU of vitamin A, 100,000 IU of vitamin D and 0.1 g of vitamin E.
^bContained per kilogram of premix: 180 g Ca, 90 g P, 20 g Mg, 60 g Na, 2 g Mn, 3 g Fe, 0.3 g Cu, 3 g Zn, 0.1 g Co, 0.1 g Se, 0.1 g I, 3 g antioxidant.

Sampling

Diets were sampled weekly and stored after oven drying for subsequent chemical analysis. For serum harvesting, blood samples were collected from each animal by venipuncture without anticoagulant (vena jugularis externa) on days 0, 28 and 56 (2 h after the morning meal) and were stored at −20°C, until analysed. Whole blood was centrifuged at 1760×g for 15 min at 4°C. For blood plasma harvesting, blood samples were collected on day 56 (2 h after the morning meal) via jugular venipuncture in Ethylenediaminetetraacetic acid (EDTA) tubes and then plasma harvested (by centrifuging method similar to serum). On days 0 and 28, serum samples were only used to determine the Cu and Zn concentrations; however, it was used for the analysis of urea nitrogen, cholesterol, concentrations of Zn and Cu on day 56. At slaughter (on day 59 of the study), samples of liver were taken from a subgroup of animals (three per replication or nine per treatment) to determine the Zn concentration. The whole liver samples were cut into pieces with titanium-coated scissors, minced with a cutter and deep-frozen.

Measurements

Dry matter intake was recorded by taking feed refusals at 08.00 am. Body weights were taken on days 0, 28 and 56. Standard procedures of AOAC(2000) were used for analysis of feed and refusal samples. Dry matter was obtained after drying at 105°C for 24 h. Kjeldahl method after acid hydrolysis was used for N and then, Crud protein = N×6.25 calculated. The Soxhlet method was used to obtain ether extract after extraction with petroleum ether. Neutral detergent fibre (NDF) was analysed by the method of Van Soest et al. (1991), where feed samples are refluxed for 1 h with neutral detergent solution without sodium sulphite and alpha-amylase, and with the residue dried and reported as NDF. A method of AOAC (1990) was used for analysing acid detergent fibre (ADF). Total-tract apparent digestibility of DM was determined using acid-insoluble ash (Van Keulen and Young 1977) as an internal digestibility marker. Sampling and analyses of faeces were done as described by Hristov and Ropp (2003). Plasma total protein, plasma albumin, plasma alkaline phosphatase (ALP), serum urea nitrogen and serum cholesterol were determined using a Roche Cobas Mira Plus Chemistry Analyzer. Analyses of Zn, Cu, Fe, Mo, Mn, Ca and Mg were done by using atomic absorption spectrophotometry (Perkin-Elmer, ICP-OES–Optima 8000 Spectrometer), P by colorimetry (Fiske and Subbarow 1925) and for S in nitric-perchloric acid by inductively coupled plasma atomic emission spectrometry (McBride and Spiers 2001).

Claw health

Clinical examinations were performed by one professional claw health manager who was trained in diagnosing claw disorders through discussions with one main author of this article (HF) and a veterinarian during claw assessment. LN prevalence was defined as the proportion of animals with the outcome of a given disorder at claw assessing date (every two weeks). For this examination, animals were restrained and its feet were thoroughly washed and trimmed for complete exposure of the lesions. At the time of visit, all cows of the farm were evaluated for their LN status using a 5-point LN score as described by Sprecher et al. (1997). During the procedure of claw assessment, front and hind claws were examined for the presence of claw disorders as described by Bielfeldt et al. (2005).

Statistical analysis

Data analysis was performed by SAS 9.1. Data were performed by ANOVA using the general linear model (GLM) procedure of SAS. Least squares means with a significant F-test (p<0.05) were compared using PDIFF of SAS. Young bulls were randomly grouped in six pens (16–19 animals per pen). Pen was used as the experimental unit with three replications. Statistical analysis of serum Cu and serum Zn concentrations were analysed as repeated measures using the mixed procedure of SAS as described by Littell et al. (1998). Animal within treatment was used as a random error term. The model included Zn, time and Zn×?time interaction. Initial values for serum Cu and serum Zn concentrations were used as a covariant in the repeated measures analysis.

Claw disorder complexes and LN were analysed as binary traits; ‘non-lame’ animals were distinguished from ‘sever lame’ animal with score >3. All animals were tested for the presence of LN and of each claw disorder with Logistic Regression Analysis. The model was run stepwise forward with probability for entry in the model p<0.05 and Zn-supplemented group used as reference. Independent variables with (p>0.10) were removed from the final model by stepwise backward elimination. The significance level was set at p<0.05.

Results

Performance and DM digestibility

Dry matter intake was not different between treatments on days 0–28 and 28–56 (Table 2). Average daily gain (p<0.01), G:F (p<0.001) and DM digestibility (p<0.001) were decreased by supplementation of Zn.

Table 2. Least squares means and standard errors of growth performance and DM digestibility for young Holstein bulls fed different diets.^a

Variable	Control	Zn	P value
DMI (kg/d)
Day 0–28	9.92	9.72	0.72
Day 28–56	10.94	10.65	0.97
Day 0–56	10.43	10.19	0.95
ADG (kg)
Day 0–28	1.46	1.18	<0.01
Day 28–56	1.29	1.10	<0.01
day 0–56	1.37	1.14	<0.01
G:F
Day 0–28	0.14	0.12	<0.001
Day 28–56	0.11	0.10	<0.001
Day 0–56	0.13	0.11	<0.001
DM digestibility (%)	69.75	58.07	<0.001
^aDiets included (1) the basal diet (control); (2) basal diet plus 150 mg of Zn/kg of DM as ZnSO4.

Metabolic profile

There was difference between treatments on concentration of serum cholesterol on day 56 (p<0.01; Table 3). There was no difference between treatments on concentration of serum Zn and Cu on days 0 and 28. ALP was affected by adding Zn (p<0.001). The Zn concentration of liver was increased by Zn supplementation (p<0.05) and serum Zn tended to increase on day 56 (p=0.06). Serum urea nitrogen also increased with Zn supplementation (p<0.01).

Table 3. Least squares means and standard errors of metabolic profile for young Holstein bulls fed different diets containing a Zn supplementation of 0 or 150 ppm.

Variable	Control	Zn	P value
Plasma total protein (mg/100 ml)	4.9	5.5	<0.01
Plasma albumin (mg/100 ml)	2.9	3.2	<0.05
Plasma ALP (U/L)	97.6	107.3	<0.001
Serum cholesterol (mg/100 ml)	97.7	91.7	<0.01
Serum urea nitrogen (mg/100 ml)	11.8	13.2	<0.01
Serum Zn (µg/100 ml)
Day 0	89.2	90.8	0.85
Day 28	83	101	0.1
Day 56	147.8	194.4	0.06
Serum Cu (µg/100 ml)
Day 0	94	91.4	0.97
Day 28	92.6	108.6	0.35
Day 56	117	127.2	0.6
Liver Zn (mg/kg DM)	164	183.5	<0.05
ALP, alkaline phosphatase.

Also, the concentration of Zn in serum of young bulls was affected by time (p<0.01; Table 4). The concentration of Cu in serum of young bulls was affected by time (p<0.01) and interaction between Zn×time (p<0.05).

Table 4. Probabilities of main effects and interactions on serum Zn and Cu of young Holstein bulls.

Variables	Zn	Time	Time×Zn
Serum Zn	0.89	<0.01	0.65
Serum Cu	0.19	<0.01	<0.05

Claw health

The prevalence of LN was 19.8%. LN was most frequently observed in the control group (28% in this group; OR = 2.7).

Discussion

Performance and DM digestibility

Results of the present study indicated that high levels of Zn supplementation (150 ppm Zn as ZnSO₄) had negative effects on ADG and G:F (Table 2). When Zn supplementation (200 ppm Zn as ZnSO₄) was added to the diets of steers, weight gain was similar to the control group (Huerta et al. 2002). In this study, DMI did not differ between treatments throughout the experiment. However, Galyean et al. (1995) reported that DMI was less by steers fed the control diet (supplemented with 30 mg/kg of Zn from ZnO) than by steers fed the other dietary treatments (supplemented with 70 mg/kg of Zn from Zn sulphate and Zn methionine) and also, observed no differences for ADG or feed efficiency among steers fed control or Zn sources diets. The opposite results may be possibly related to environmental differences or experimental conditions.

The iron requirement is approximately 50 mg/kg diet in beef cattle that this level is adequate to support growth in young calves (Bernier et al. 1984; NRC 2000). Iron deficiency was resulted to low performance and high susceptibility to disease (Möllerberg et al. 1975). Marginal Fe(30.2 ppm) in the basal diet with Zn(150 ppm) might negatively affect the growth, but clinical signs of a lack of Fe were not shown.

Mandal et al. (2007) indicated that a diet containing about 32.5 mg Zn/kg DM was adequate to support normal growth and digestibility in bulls. Supplementation of Zn decreased G:F between days 28 and 56 while depressing DM digestibility. Froetschel et al. (1990) had shown that Zn supplementation (1142 ppm Zn as Zinc sulphate) reduced post-ruminal passage of bacterial amino acid (AA) to an extent that the net output of post-ruminal AA as a percentage of AA intake was reduced. This may partly explain the poor performance of young bulls fed 150 ppm zinc in the form of zinc sulphate in the present study. On the other hand, marginal Cu in control diet might negatively affect the ADG and G:F, and it was intensified by high levels of Zn supplementation where the level of Zn was five times higher than NRC recommendations (150 ppm vs. 30 ppm). Ration in this experiment contained 44.8, 4.7, 30.2 and 59 ppm of Zn, Cu, Fe and Mn before supplementation with Zn (150 ppm). In a study, apparent digestibility of dry matter, ADG and G:F decreased when the ration contained 102, 6 and 51 ppm of Zn, Cu and Mn in Holstein bull calves during a 10-wk feeding trial; However, ADG and G:F increased when Zn without Mn was added to the basal diet, and growth performance decreased when Mn without Zn was added to the basal diet (Ivan and Grieve 1975). Also, they indicated that the apparent digestion coefficients of N and gross energy were lower in the ration with Zn and Mn than control group, Zn-received animals and Mn-received group. Therefore, we expected that the excessive levels of Zn and Mn might negatively affect the growth performance and DM digestibility in Zn-received young bulls rather than the marginal levels of Cu or Fe in the basal diet. Nunnery et al. (2007) reported that DMI and ADG did not differ among control beef heifers and heifers in the three supplemental Zn treatments (with 75 mg of supplemental Zn/kg of DM from Zn sulphate, Zn methionine or Zn propionate); however, overall G:F tended to be less for control heifers than for heifers in the three supplemental Zn treatments in the finishing period. Therefore, growth performance will increase by Zn supplementation if sufficient trace mineral is supplied.

When one in vivo metabolism experiment was used to examine the effects of supplemental Zn on ruminal parameters, digestion and DMI by heifers fed low-quality prairie hay supplemented with urea, it was determined that a concentration of 250 ppm may decrease the likelihood of urea toxicity and may increase energetic efficiency of ruminal fermentation (by increasing the proportion of propionate in ruminal volatile fatty acid or VFA); however, 470 ppm added Zn tended to decrease intake of digestible DM, primarily due to a trend for reduced digestibility with 470 ppm supplemental Zn (Arelovich et al. 2000). Also, high dietary concentration (1142 mg Zn per kg) of inorganic Zn has also increased molar proportion of propionate in a previous study (Froetschel et al. 1990). Also, they indicated that the effects of zinc on ruminal AA digestion may be more closely related to an interaction of zinc with dietary CP rather than to an effect of Zn on ruminal microbial populations. Altogether, the decrease in growth performance by supplemented Zn might be related to the decrease in DM digestibility or alteration of ruminal fermentation efficiency. However, in most studies with various species, no adverse physiological effects were observed with less than 600 ppm supplemental Zn. In general, all-plant diets should be supplemented with Zn because plant sources have lower zinc than animal protein sources. Phytate present in high concentrations in soybean meal and other plant proteins tie up Zn in the gastrointestinal tract, thereby increasing the dietary requirement (McDowell 2003). Zinc requirements may increase when stress increases in young bulls or animals suffer illness, especially in damp situations. Plasma Zn, as part of an acute phase response, is initially reduced by infection (Wellinghausen and Rink 1998), only to become elevated within a few days. Serum Zn is also decreased by hyperthermal stress and ketosis in cows and is increased in cows with mastitis and in older cows (Wegner et al. 1973).

Metabolic profile

In the present study, the Zn concentration of liver was increased by Zn supplementation, and serum Zn tended to increase on day 56 (Table 3). Also, the concentration of Zn in the serum of animals was affected by time (Table 4). The concentration of Cu in the serum of young bulls was affected by time and interaction between Zn×time. Also, ALP did not differ. Wright and Spears (2004) indicated that the addition of 20 mg of Zn/kg of DM to a control diet (with 28 mg of Zn/kg of DM) did not increase plasma Zn concentration or ALP activity. In a previous study, the concentration of Zn in the liver of steers was affected by time and the level of dietary Zn. Steers supplemented with 200 ppm Zn had higher concentrations of Zn in liver at days 59 and 134 of the experiment than steers fed the control diet (Huerta et al. 2002). Ivan and Grieve (1975) reported that the addition of Zn and Cu to the ration of bull calves did not affect concentrations of Zn in liver tissue, although Cu appeared to result in a slight decrease. The addition of Mn, and particularly in combination with Zn and Cu, appeared to increase the concentration of Zn in liver tissue. Malcolm-Callis et al. (2000) reported no differences in serum Zn concentrations measured on day 28 or 112 among steers supplemented with Zn sulphate, Zn polysaccharide or a Zn AA complex; and Spears and Kegley (2002) reported no difference in serum Zn concentrations between steers supplemented with Zn proteinate or ZnO. Huerta et al. (2002) reported that high dietary Zn (200 ppm) appears to depress serum Cu concentration. However, Zn levels did not affect the serum Cu concentration, which may be due to control of serum Zn concentrations by means of complex mechanisms in body. In this study, serum urea nitrogen also increased with Zn supplementation. Huerta et al. (2002) indicated that serum urea nitrogen did not differ when feedlot heifers were supplemented with 200 ppm Zn.

In a study, urinary Cu and Zn excretion decreased during stress and also, Zn retention decreased and Cu retention became negative when no supplemental mineral (copper and zinc) was fed to crossbred steer calves (Nockels et al. 1993).

In another experiment it was indicated that there was no alteration in immune response due to Zn sulphate supplementation in bulls after 150 days of feeding, where male crossbred cattles were supplemented with 35 mg Zn/kg DM plus basal diet with 32.5 mg Zn/kg DM (Mandal et al. 2007). High dietary Zn can accentuate borderline deficiencies of other element, including Fe and Cu (McDowell 2003) and it may negatively affect the immune system or metabolism or performance.

Claw health

The prevalence of LN was high in this experiment because of high humidity. Certain environmental, genetic and dietary factors may influence animal performance response to trace mineral supplementation. Therefore, we chose a damp situation to evaluate the effects of Zn supplementation when this trace mineral was added five times higher than NRC recommendation (30 mg/kg DM Zn) for LN decreasing. A 2007 survey of consulting nutritionists revealed that the most common Zn concentration in finishing diets were 100 mg/kg DM (Vasconcelos and Galyean 2007). This same survey listed maximum concentration in finishing diets as 212.5 mg/kg DM Zn. In the present experiment, we totally used 194.8 mg/kg DM Zn (the basal diet + Zn supplementation) to determine Zn effects in stress conditions.

Lameness was most frequently observed in the control group. The percentage of affected animals was 1.88% for sole disorders (SD), 1.88% for white line disorders (WD), 0% for heel erosion (HE) and 16.04% for skin and inter-digital space disorders (ID). The prevalence of ID was the greatest cause of LN in the present study (80.9% of whole LN) and was higher in control than another group (47.6% from 80.9%). Statistically, the prevalence of WD, HE and SD were not different; but, the prevalence of LN was totally significant (p<0.05; Table 5). The prevalence of LN was higher in control group than ZnSO₄-supplemented young bulls (OR = 2.7; Table 6) and also, the odds ratio of ID was 1.75; however, the prevalence of ID was not significant (p = 0.29). Zinc supplementation for stressed cattle enhanced the recovery rate in infectious bovine rhinotracheitis virus-stressed cattle (Chirase et al. 1991) and supplementing it to dairy cows during lactation resulted in fewer infections of mammary gland (Spain et al. 1993). Also, feeding Zinc can improve hoof quality scores in fattening bulls (Kessler et al. 2003). Hatfield et al. (2002) demonstrated that high levels of supplemental Zn may have negative effects on humoral immune function in ewes. However, Galyean et al. (1995), Salyer et al. (2004) and Nunnery et al. (2007) reported no differences in morbidity of newly received beef cattle as affected by supplemental Zn source. The high prevalence of skin and ID in Zn-supplemented animals, where the prevalence of WD, HE and SD, were low in this group, might be due to the imbalance of mineral in the basal diet, especially Mn, Cu and Fe.

Table 5. Probabilities of main effects on the presence of claw disorders of young Holstein bulls.

Lameness	Sole disorder	White line disorder	Inter-digital disorder
<0.05	0.94	0.94	0.29

Table 6. Associations between treatments and the presence of claw disorders in young Holstein bulls.

	Lameness		Sole disorder		White line disorder		Inter-digital disorder
	OR	CLL–CLU	OR	CLL–CLU	OR	CLL–CLU	OR	CLL–CLU
Control	2.7	0.99–7.43	>999.9	<0.001– > 999.9	>999.9	<0.001– > 999.9	1.75	0.61–5.01
Zn	1		1		1		1
OR, odds ratio; CLL, lower confidence limit; CLU, upper confidence limit; p =0.05.

Nevertheless, any preventive way has not been reported since digital dermatitis or other skin and ID were known. However, Fagari-Nobijari et al. (2010) reported that dietary chlortetracycline was better than Zn supplementation for controlling of LN. Topical treatment with chlortetracycline resulted in resolution from digital dermatitis (Holzhauer et al. 2008). As numerically, Zn could decrease ID prevalence. It is important to find a suitable alternative to antibiotics such as chlortetracycline; however, the controlling of vitamins and mineral levels in finishing diets may help to decrease diseases. In the present study, wet floor, where this experiment had been done, was the most important cause of skin and ID, especially digital dermatitis that increased the prevalence of LN.

Conclusion

Zinc supplementation (150 mg of Zn/kg of DM as ZnSO₄) in finishing diets decreased growth performance of young Holstein bulls; however, Zn supplementation might have positive effects on control of LN. The basic reason of growth decrease was unclear. High levels of supplemental Zn may do so with risk of adversely affecting the animal's performance when Cu level in the basal diet of young bulls was less than NRC recommendations; however, copper status in serum was not changed which might be related to the controlling of Zn status in blood. Therefore, if the levels of minerals such as Mn, Cu and Fe are controlled in young bull's diets, animal producers are able to use Zn for controlling LN rather than using antibiotics. Totally, further research is needed to determine the proper level of Zn for controlling LN and to increase the growth performance in young bulls when the levels of minerals are balanced.