Effects of dietary supplementation of chelated water-soluble mineral mixture on growth performance, nutrient digestibility, blood profiles and faecal micro flora in weanling pigs

ABSTRACT

A total of 105 [(Landrace × Yorkshire) × Duroc] pigs with an initial body weight (BW) of 7 ± 1.58 kg were used in a 5-week experiment in two phases to test the efficacy of supplementation of chelated mineral mixture on piglet performance. Pigs were randomly allocated to one of three treatments [five pigs per pen (three barrows and two gilts); seven pens per treatment]. Treatments consisted of: CON; basal diet, TRT1; basal diet + 0.89% chelated mineral mixture and TRT2; basal diet + 0.55% chelated mineral mixture. Pigs fed TRT1 and TRT2 had increased (P < .05) gain/feed ratio during the first phase and overall and had greater (P < .05) average daily gain (ADG) during the second phase compared with CON. Pigs fed TRT1 had greater (P < .05) ADG overall and had higher (P < .05) dry matter (DM) digestibility during the first phase. On day 35, the concentrations of Fe and Cu in blood serum were higher (P < .05) in TRT1 and TRT2 but Ca concentration increased (P < .05) and faecal Escherichia coli counts reduced in TRT1 compared with CON. In conclusion, our results indicated that chelated mineral mixture have beneficial effects in pigs.

KEYWORDS:

• Chelated mineral mixture
• growth performance
• micro flora
• weanling pigs

1. Introduction

With growing restriction on the use of antibiotics as a growth promoter due to bacterial resistance issues as well as increasing public concern about drug residue in meat products, an intensive amount of research has been focused to find an alternative to antibiotics to maintain swine health and performance. The most widely investigated alternatives include plant extract, acidifiers, prebiotics, probiotics and neutraceutical such as copper and zinc. The effectiveness of the use of minerals as a replacer to antibiotics for improved performance depends on their bioavailability to animals. Thus, it is essential to know the relative bioavailability of these minerals from feed ingredients or complete diet in order to determine how efficiently an animal utilizes dietary mineral elements.

Research has demonstrated that bioavailability of minerals varies considerably between sources. Organic mineral complexes are said to be more bioavailable than inorganic salts because inorganic salts are reported to be rapidly dissociated and become free to interact with an antagonist, thereby leading to loss of mineral prior to absorption by animals (Henry et al. 1992; Ward et al. 1996). However, an organic mineral complex formed by chelation of minerals with organic acids, amino acids or peptides prevents the minerals from interacting with the antagonist because the minerals are bound to organic ligands through covalent bonds, thereby improving the bioavailability of minerals (Ward et al. 1996). Thus, contrasting results are shown with the studies on bioavailability of organic and inorganic minerals. For instance, an organic mineral complex formed by chelation of Fe is reported to have 125–185% bioavailability compared with an inorganic source of Fe such as ferrous sulphate (Henry & Miller 1995). Similarly, Coffey et al. (1994) noted improvement in the performance of piglets when copper lysine was used compared with copper sulphate. Zhang et al. (2013) demonstrated that 0.1% chelate copper and zinc improved growth performance and nutrient digestibility in weanling pigs. Thus, researches on dietary chelated minerals have gained considerable attention in the past few years.

The present study was conducted to evaluate the effect of novel composition of mineral water containing a mixture of macro and micro mineral chelates with natural extract for their potential to replace antibiotics as growth promoter for use in swine production.

2. Materials and methods

The experiment was conducted at the Experimental Unit of the Dankook University (Anseodong, Cheonan, Choongnam, Korea). The protocol for the current experiment was approved by the Animal Care and Use Committee of Dankook University.

2.1. Source of mineral mixture

The chelated liquid mineral mixture used in the current study was provided by the commercial company (Jino Biotech, Dongnam-gu, Cheonan-si, South Korea). According to the supplier's information, inorganic mineral ions were extracted effectively in ceramics (natural mineral) and the extractions of inorganic ions were maximized using hydrodynamic cavitation as mentioned by Zhou et al. (1997). Chelated mineral was produced by mixing the ionized minerals with natural extract. This natural extract was obtained through Bacillus fermentation of plants such as wild grapes and prickly pear. This mineral mixture which was in impure form was purified using ion exchange membrane filters having micro pores. The composition of the mineral mixture is shown in Table 1.

Table 1. Chelated mineral mixture composition.

Items	Chelated mineral 1	Chelated mineral 2
Mineral elements, mg/L
K	102.04	66.80
Na	1408.06	1509.20
P	101.50	109.19
Ca	2461.27	1021.10
Mg	1909.62	309.48
Fe	1049.41	854.76
Al	1133.21	1194.01
Mn	138.23	152.35
Si	183.39	136.41
Zn	30.65	5.79
Cu	80.84	65.10
Zr	5.31	4.71
Ti	24.23	27.03
Ce	2.63	2.09
Nd	1.22	1.65
La	1.19	1.01
V	131.48	–
Se	51.29	1.18
Co	42.09	42.09
Ge	20.08	0.43
Mo	0.55	0.78
Ba	12.62	14.16
Cr	0.24	0.25
Ni	19.36	20.25
Br	0.24	0.25
Nb	1.23	–
Ag	4.67	–
I	3.04	–
Cl	0.22	0.25
W	6.78	–
Total	8926.69^a	5540.32^a

Note: Chelated mineral 1 containing 0.89% of the mineral mixture was used in TRT1 and chelated mineral 2 containing 0.55% of the mineral mixture was used in TRT2.

^aMineral mixture 0.89% or 0.55% was administered to pigs by diluting 10 ml of each of the mineral mixture chelates in 20 L tap water.

2.2. Experimental design, animals, housing and diets

A total of 105 cross-bred [(Landrace × Yorkshire) × Duroc] weanling pigs were used in a 5-week trial. Pigs were randomly allocated into one of three treatments (seven replications with five pigs per pen) in a randomly complete block design. Treatment consisted of (1) CON, basal diet and normal tap water, (2) TRT1, basal diet + 10 ml of 0.89% chelated water-soluble mineral mixture in 20 L tap water and (3) TRT2, basal diet + 10 ml of 0.55% chelated water-soluble mineral mixture in 20 L tap water. The chelated water-soluble mineral mixture was administered to pigs in a bucket one or two times per day, and the water was changed every day. The diets used in this experiment were formulated to meet or exceed NRC (2012) requirements (Table 2). Pigs were housed in an environmental controlled, slatted-floor facility and mechanical ventilation system. The temperature of the room was maintained at approximately 30°C for the first week of the experiment, after which it was reduced by 1°C per week over the next 4 weeks. The reason for subsequent reduction of temperature by 1°C per week is due to the fact that as pigs grow, suitable temperature is required for metabolic comfort. If the temperature is above the metabolic comfort of pigs, it will reduce their appetite as a response to increased body heat that cannot be dissipated to the environment. Each pen was equipped with a self-feeder and a nipple waterer to allow ad libitum access to feed and water throughout the experimental period.

Table 2. Feed compositions of basal diet (as-fed basis).

Item	Phase 1 (1–14 days)	Phase 2 (15–35 days)
Ingredient, %
Corn	11.91	28.14
Extruded corn	28.05	21.91
Soybean meal (48% CP)	29.8	36.5
Soy oil	3.7	4.5
Lactose	8.0	–
Whey	15.0	5.0
Monocalcium phosphate	1.2	1.4
L-Lys HCl (78%)	0.54	0.53
DL-Met (50%)	0.14	0.31
L-Thr (89%)	0.22	0.21
Vitamin premix^a	0.1	0.1
Mineral premix^b	0.2	0.2
Limestone	0.94	0.9
Salt	0.2	0.3
Calculated composition
Metabolizable energy, kcal/kg	3700	3650
Analysed composition,%
Crude protein	21.7	20.5
Lysine	1.4	1.35
Calcium	0.65	0.62
Total phosphorous	0.52	0.5
Methionine	0.60	0.55
Crude fibre	2.01	2.55

^aProvided per kg of complete diet: vitamin A, 11,025 IU; vitamin D₃, 1103 IU; vitamin E, 44 IU; vitamin K, 4.4 mg; riboflavin, 8.3 mg; niacin, 50 mg; thiamine, 4 mg; d-pantothenic, 29 mg; choline, 166 mg; and vitamin B₁₂, 33 μg.

^bProvided per kg of complete diet: Cu (as CuSO₄•5H₂O), 12 mg; Zn (as ZnSO₄), 85 mg; Mn (as MnO₂), 8 mg; I (as KI), 0.28 mg; and Se (as Na₂SeO₃•5H₂O), 0.15 mg.

2.3. Chemical analysis

Samples of diets were analysed using standard methods (AOAC 2000) for nitrogen (N; method 968.06), crude fibre (method 962.09). Calcium (method 984.01) and phosphorus (method 965.17) contents were determined according to the AOAC (1995). The individual amino acid composition was measured using an amino acid analyser (Beckman 6300, Beckman Coulter, Inc., Fullerton, CA, USA) after 24 h of 6 N HCl hydrolysis at 110°C (AOAC 2000). To determine the methionine levels, the samples were oxidized with performic acid overnight. Nitrogen was determined using a Kjectec 2300 Nitrogen Analyser (Foss Tecator AB, Hoeganaes, Sweden). The gross energy was determined by measuring the heat of combustion in the samples using a Parr 6100 oxygen bomb calorimeter (Parr Instrument Co., Moline, IL, USA). The calculated metabolizable energy during the first and second phases was 3700 and 3650 kcal/kg, respectively. The analysed crude protein content of the experimental diet during the first and second phases was 21.7% and 20.5%, respectively.

2.4. Experimental procedure, sampling and assay

Body weight (BW) and feed consumption were measured at the beginning and on the second and fifth weeks of the experimental period to monitor the average daily gain (ADG), average daily feed intake (ADFI) and gain:feed ratio (G/F). Feed consumption was determined on a pen basis during the experiment. Chromic oxide was added to the diet as an indigestible marker at a concentration of 0.20% for 7 days prior to faecal collection for calculation of dry matter (DM), nitrogen (N), gross energy (E), calcium (Ca) and phosphorus (P) digestibility. Faecal samples were collected from 2 pigs in each pen (1 gilt and 1 barrow; 14 pigs per treatment) via rectal massage at days 12, 13 and 14 (first phase) and days 33, 34 and 35 (second phase). All faeces samples were stored immediately at −20°C until analysis. Faecal samples were dried at 70°C for 72 h and finely ground to pass through a 1-mm screen. All of the faecal samples were then analysed for DM and N following the procedures outlined by the AOAC (2000). Chromium was analysed using UV absorption spectrophotometry (Shimadzu, UV-1201, Kyoto, Japan) and nitrogen was determined using a Kjeltec 2300 Analyser (Foss Tecator AB, Hoeganaes, Sweden). Gross energy was determined by measuring the heat of combustion in the samples using a Parr 6100 oxygen bomb calorimeter (Parr instrument Co., Moline, IL, USA).

The apparent total tract digestibility was calculated according to the method described by Fenton and Fenton (1979) using the following formula: digestibility (%) = {1−[(Nf × Cd)/(Nd × Cf)]} × 100, where Nf = nutrient concentration in faeces (% DM), Nd = nutrient concentration in diet (% DM), Cd = chromium concentration in diet (% DM), and Cf = chromium concentration in faeces (% DM).

For blood profile, 2 pigs from each pen (n = 14 per treatment) were randomly selected and bled via jugular venipuncture using a sterile needle at days 14 and 35. Blood samples of the pig were collected into vacuum tubes containing K₃EDTA (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) to obtain whole blood. The red blood cell (RBC), white blood cell (WBC) concentration and lymphocyte percentage of the whole blood samples were determined using an automatic blood analyser (ADVIA 120, Bayer, Tarrytown, NY, USA). Samples for serum analysis were then centrifuged at 3000 × g for 15 min at 4°C and serum was stored at −20°C until analysis. Calcium, phosphorus, iron and copper were determined using an automatic biochemistry blood analyser (HITACHI 747, Hitachi, Tokyo, Japan).

For microbial analysis, fresh faeces was collected directly via massaging the rectum from 2 pigs per pen (n = 14 pigs per treatment) at days 14 and 35 of the experiment and, respectively, pooled and placed on ice for transportation to the laboratory. The composite faecal sample (1 g) from each pig was diluted with 9 mL of 1% peptone broth (Becton, Dickinson and Co., Franklin Lakes, NJ) and then homogenized. Viable counts of bacteria in the faecal samples were studied by plating serial 10-fold dilutions in anaerobic diluents before inoculation on to Petri dishes of sterile agar. Salmonella and Escherichia coli present in the fresh faecal samples were enumerated. The selective medium for Salmonella was salmonella-shigella agar (Difco, USA) and for E. coli was Mac Conkey agar (Difco, USA). After inoculation, the entire dish was inverted and incubated anaerobically at 37°C for 48 h. The colony counts were then enumerated and results are presented as log₁₀-transformed data.

2.5. Statistical analyses

Data were analysed by ANOVA using the General Linear Model procedure of SAS (SAS Institute 1996), with pen being defined as the experimental unit. Differences among treatments were separated by Tukey's test. Variability in the data is expressed as pooled standard error and a probability level of P < .05 was considered to be statistically significant and P < .10 being considered as a tendency.

3. Results

3.1. Growth performance and nutrient digestibility

During days 1–14, pigs fed TRT1- and TRT2-improved (P < .05) feed efficiency compared with those fed CON (Table 3). Pigs fed TRT1 and TRT2 had greater (P < .05) ADG on days 15–35 compared with CON. Overall, pigs fed TRT1 and TRT2 had greater (P < .05) feed efficiency and pigs fed TRT1 had greater ADG (P < .05) compared with CON. The effect of the chelated mineral mixture on nutrient digestibility is presented in Table 4. Pigs fed TRT1 diet had greater (P < .05) DM digestibility compared with those fed CON diet during the first phase but DM digestibility was not affected during second phase of the experiment. No difference (P > .05) was observed on nitrogen, gross energy, Ca and P digestibility at both phases of the experiment.

Table 3. The effects of chelated water-soluble mineral supplementation on growth performance in weanling pigs

Items	CON^a	TRT1^a	TRT2^a	SE^b	P-value
Phase 1 (1–14 days)
ADG, g	332	352	341	9	.29
ADFI, g	412	418	409	9	.78
G/F	0.806^d	0.842^c	0.834^c	0.009	.01
Phase 2 (15–35 days)
ADG, g	535^d	555^c	550^c	8	.22
ADFI, g	805	799	794	7	.55
G/F	0.665	0.695	0.693	0.009	.07
Overall (1–35 days)
ADG, g	454^d	474^c	466^c,d	6	.09
ADFI, g	648	647	640	7	.67
G/F	0.701^d	0.733^c	0.728^c	0.006	.007

^aAbbreviation: (1) CON, basal diet and normal tap water, (2) TRT1, basal diet + 10 ml of 0.89% chelated water-soluble mineral mixture in 20 L tap water and (3) TRT2, basal diet + 10 ml of 0.55% chelated water-soluble mineral mixture in 20 L tap water.

^bStandard error (pooled).

^c,dMeans in the same row with different superscripts differ (P < .05).

Table 4. The effects of chelated water-soluble mineral supplementation on nutrient digestibility in weanling pigs.

Items,%	CON^a	TRT1^a	TRT2^a	SE^b	P-value
14 day
DM	82.63^d	85.64^c	84.25^c,d	0.54	.05
N	81.27	82.63	82.51	0.63	.26
Gross energy	82.65	84.55	83.24	0.60	.10
Ca	62.25	65	63.09	1.20	.28
P	47.75	43.31	44.84	3.76	.70
35 day
DM	80.96	81.8	81.38	1.06	.85
N	78.71	80.26	79.91	1.15	.61
Gross energy	79.61	81.11	80.48	1.09	.63
Ca	56.3	57.47	56.89	2.45	.94
P	45.14	40.81	43.81	3.89	.72

^aAbbreviation: (1) CON, basal diet and normal tap water, (2) TRT1, basal diet + 10 ml of 0.89% chelated water-soluble mineral mixture in 20 L tap water and (3) TRT2, basal diet + 10 ml of 0.55% chelated water-soluble mineral mixture in 20 L tap water.

^bStandard error (pooled).

^c,dMeans in the same row with different superscripts differ (P < .05).

3.2. Blood characteristics

The lymphocyte, WBC, RBC and minerals such as P and Mg were not influenced (P > .05) by the chelated mineral mixture supplement on the second and fifth weeks of the experiment. Ca and Fe concentrations were not influenced (P > .05) by the chelated mineral mixture supplement on day14. However, dietary TRT1 and TRT2 supplement led to higher (P < .05) Ca and Fe concentrations compared with the CON group on day 35. Pigs fed TRT1 and TRT2 diet showed greater (P < .05) Cu concentration in blood serum compared with CON on days 14 and 35 (Table 5).

Table 5. The effects of chelated water-soluble mineral supplementation on blood profiles in weanling pigs.

Items	CON^a	TRT1^a	TRT2^a	SE^b	P-value
Lymphocyte, %
14 day	50.3	50.42	52.92	4.60	.90
35 day	52.12	53.18	56.58	4.01	.72
RBC, 10^6/µl
14 day	6.1	6.15	6.17	0.15	.94
35 day	6.12	6.19	6.26	0.08	.50
WBC, 10^3/µl
14 day	16.83	17.12	17.63	1.05	.86
35 day	17.08	18.55	18.19	1.03	.59
Calcium, mg/dL
14 day	10.9	13.8	13.64	1.08	.16
35 day	12.48^d	17.60^c	15.25^c,d	1.42	.02
P, mg/dL
14 day	7.32	8.98	9.44	0.82	.21
35 day	8.80	12.68	11.84	0.97	.05
Mg, mg/dL
14 day	1.8	1.9	1.96	0.17	.79
35 day	2.1	2.16	2.3	0.27	.87
Fe, g/dL
14 day	57.8	65	69.6	10.03	.71
35 day	58.50^d	96.50^c	92.25^c	9.47	.02
Cu, g/dL
14 day	55.08^d	94.53^c	104.17^c	8.15	.006
35 day	68.34^d	121.96^c	109.12^c	12.01	.03

^aAbbreviation: (1) CON, basal diet and normal tap water, (2) TRT1, basal diet + 10 ml of 0.89% chelated water-soluble mineral mixture in 20 L tap water and (3) TRT2, basal diet + 10 ml of 0.55% chelated water-soluble mineral mixture in 20 L tap water.

^bStandard error (pooled).

^c,dMeans in the same row with different superscripts differ (P < .05).

3.3. Faecal microflora

At the end of the experiment, faecal E. coli counts were reduced (P < .05) in the TRT1 and tended to be reduced in TRT2 (Table 6). No difference (P > .05) was observed on the Salmonella counts during the whole experiment.

Table 6. The effects of chelated water-soluble mineral supplementation on faecal microflora in weanling pigs.

Items, log10 cfu/g	CON^a	TRT1^a	TRT2^a	SE^b	P-value
14 day
Salmonella	2.39	2.23	2.21	0.05	.10
E. coli	6.59	6.10	6.31	0.08	.35
35 day
Salmonella	2.44	2.24	2.17	0.08	.12
E. coli	6.54^c	6.20^d	6.36^c,d	0.07	.02

^aAbbreviation: (1) CON, basal diet and normal tap water, (2) TRT1, basal diet + 10 ml of 0.89% chelated water-soluble mineral mixture in 20 L tap water and (3) TRT2, basal diet + 10 ml of 0.55% chelated water-soluble mineral mixture in 20 L tap water.

^bStandard error (pooled).

^c,dMeans in the same row with different superscripts differ (P < .05).

4. Discussion

The source of macro and micro minerals for piglets is from inherent and supplemental minerals provided in nursery diets. During the period of transition from sows’ milk to dry feed, the newly weaned pigs are faced with challenges of adapting to a dry diet with a vastly different composition due to which the morphology and functions of the gastrointestinal tract are remarkably impaired in weanling piglets (Moeser et al. 2007). Bioavailability of both macro and micro minerals is essential for better growth performances and health status of the weaned piglets. According to this experiment, with the supplementation of diets with the addition of different concentrations of water-soluble chelated organic mineral mixture complex, there was no improvement in the growth performance except for feed efficiency during the first phase (days 1–14). The probable reason for the lack of response could be the greater mineral status of pigs at weaning. However, ADG was improved in the second phase (days 15–35) which indicated that the minerals were bioavailable for absorption and that the dosage of mineral supplementation was suitable for the growth of pigs. However, nutrient (N, gross energy, Ca and P) digestibility was not much influenced except for DM. A report by Paik (2001) demonstrated that methionine-Zn chelate at 100 and 200 ppm and zinc oxide at 200 ppm significantly improved weight gain and feed intake in weanling pigs compared with the controls (100 ppm of Zn from ZnO). The supplementation of organic trace minerals such as Cu, Fe, Zn and Mn improved nutrient digestibility in male calves (Mondal et al. 2008) and 0.1% chelate Cu and Zn supplementation improved nutrient digestibility in weanling pigs (Zhang et al. 2013). Likewise, Stanton et al. (2000) reported that organic trace mineral enhanced calf performance compared to inorganic trace minerals. In contrast, Ahola et al. (2004) reported that BW and body condition of beef cattle did not differ with organic trace minerals such as copper, zinc and manganese compared with control. The variation in result could be due to types of minerals used, age and species of animals. There are no studies to compare the effect of the chelated macro and micro mineral mixture complex on growth performance and nutrient digestibility. However, we believe that the mineral mixture complex in the present study could have interacted to positively affect growth performance and DM digestibility.

Results of blood profiles indicated that RBC, WBC and lymphocyte were not influenced by chelated mineral water treatment which is in agreement with the study by Zhang et al. (2013) except for lymphocyte. In contrast to our experiment, Zhang et al. (2013) noted an increment in lymphocyte percentage with the supplementation of 0.1% chelated Cu and Zn. In the present experiment the concentrations of Ca, Fe and Cu in the blood serum were increased and P concentration tended to increase with the supplementation of the chelated water-soluble mineral mixture, suggesting that these chelated metal ions added to water were absorbed to a certain extent by weanling pigs due to their bioavailability.

When trace minerals are fed in excess of animal requirements, more is excreted in waste because of homeostatic mechanisms that serve to regulate tissue concentrations of minerals (Spears 1996). The excreted minerals serve as environment pollutants. In this study, supplemented chelated minerals had good absorbability which means reduction in the excretion of mineral through faeces. Mondal et al. (2008) reported that absorbability of organic macro minerals was not significant compared with control and inorganic groups but maximum absorbability was seen in organic micro element-supplemented groups compared with inorganic groups and controls.

Previous studies indicated that dietary supplementation with high levels of minerals such as zinc in piglets improved gastrointestinal health and faecal micro biota due to antibacterial effect. The same antibacterial effect was noted in sows when their diet was supplemented with 250 ppm Cu sulphate resulting in a greater number of pigs born and heavier pig weaning weights over a six-parity period compared to a diet with 9 ppm Cu (Cromwell et al. 1993). When sows were fed pharmacological levels of an organic zinc amino acid complex, the nursing sows had greater integrity of the intestinal epithelium along with a better immune capability (Caine et al. 2001), indicating that the organic zinc amino complex had an antibacterial effect. In the present study, faecal E. coli counts were reduced with TRT1, indicating that the mineral water had an antibacterial effect and that the dosage of mineral in TRT1 was suitable for suppression of E. coli.

In conclusion, our results indicated that chelated mineral water containing macro and micro mineral mixture supplementation can increase growth performance, increase calcium, iron and copper concentration in blood, and decrease the faecal E. coli counts when compared with the CON treatment. Thus, supplementation of mineral chelates creates opportunities for environmental-friendly nutritional additives considering the growing restrictions on the use of antibiotics as growth promoter for improved performance and productivity.

Disclosure statement

No potential conflict of interest was reported by the authors.