Effects of silicate derived from quartz porphyry supplementation in the health of weaning to growing pigs after lipopolysaccharide challenge

ABSTRACT

The objective of this study was to investigate effects of silicate (SIL) supplementation on growth performance, nutrient digestibility, organ weight, immune traits, fecal microbiota, rectal temperature, and diarrhea score in weaning to growing pigs with challenged lipopolysaccharide (LPS). In experiment 1, a total of 72 piglets [(Landrace × Yorkshire) × Duroc] with initial body weight (BW) of 7.53 ± 0.1 kg were used in 14 weeks. Treatment groups: (1) Basal diet (CON), and (2) Basal diet + 0.1% silicate (SIL). In experiment 2, after reaching an average BW of 28.12 ± 0.79 kg at end of this experiment, 24 piglets in each treatment (12 pigs per treatment and 2 pigs per pen) were challenged with 100 μg/kg LPS. In experiment 1, SIL diets improved (P < 0.05) growth performance, crude protein (CP) digestibility and reduced (P < 0.05) NH₃ emission compared to CON diet. In experiment 2, after piglets were fed SIL diets, numbers of white blood cells (WBCs) such as WBC, neutrophil, monocyte, and lymphocyte were increased (P < 0.05). SIL diets decreased (P < 0.05) cortisol level, Escherichia coli count, diarrhea incidence, and rectal temperature compared to CON diet.

KEYWORDS:

• Silicate
• diarrhea
• immune
• Lipopolysaccharide

Introduction

The European Union has banned the use of antibiotics since 2006 because there is risk of antibiotic resistance being transmitted from animals to humans (Barton 2000; Casewell et al. 2003). Thus, many researchers are searching for alternatives to antibiotics and feeding strategies to maintain the growth performance and health of pigs. Silicates as well-known clay minerals can be used as alternatives of antibiotics, including bentonite, zeolite, kaolin (Trckova et al. 2004; Velebil 2009), montmorillonite, smectite, illite, kaolinite, and clinoptilolite (Vondruskova et al. 2010). Many researchers have reported that the supplementation of silicate can improve growth performance (Teleb et al. 2004; Acosta et al. 2005; Miles and Henry 2007), protein digestibility (Pasha et al. 2008), enzyme activities such as amino peptidase, alkaline phosphatase, and maltase (Ma and Guo 2008) and small intestinal villi (Tatar et al. 2008) in broiler and pigs. However, other researchers have shown that there is no significant difference in energy digestibility (Safaeikatouli et al. 2012) or carcass traits (Yalcin et al. 1995; Salari et al. 2006) when broilers were fed dietary silicate. Ramu et al. (1997) and Martinez et al. (2004) have reported that using dietary silicate can decrease the occurrence and severity of diarrhea in pigs. However, previous studies on dietary silicate have not investigated the effect of silicate on immunity in pigs. Recently, it has been reported that silicon dioxide can affect the immune response of animals. Yu et al. (2012) have reported that silica can decrease activities of antioxidant enzymes in the liver when mice are repeatedly exposed to an intravenous injection. Lee et al. (2013) have reported that T cells and B cells as natural killer cells in the spleen and inflammatory cytokines in the sera of mice fed silica are lower than those in the control group. However, whether silicon dioxide affects the immune response of pigs remains unclear. Injection of LPS, an outer membrane of gram-negative bacteria, is the best way to stimulate animals’ immune system and used for the experiments to check on pigs’ immunity (Wyns et al. 2015). Therefore, by injecting LPS to weaning pigs, one experiment was conducted to determine how supplementation of silicate affects pigs’ health such as immunity, stress, microbiota in feces, diarrhea and rectal temperature. Also, other experiment was performed to investigate effects of silicate supplementation on growth performance, nutrient digestibility, and organ weight in weaning to growing pigs.

Materials and methods

The experimental protocol used in this study was approved by the Animal Care and Use Committee (ACUC) of Chungbuk National University.

Compositions of silicate

Silicate used in the experiment as a feed supplement was obtained from a commercial company (TNC Co Ltd, Daejeon, South Korea). It was composed of silicon dioxide (SiO₂, 66.8%), sodium oxide (Na₂O, 26.9%), phosphorus trioxide (P₂O₃, 5.06%), and aluminum oxide (Al₂O₃, 0.05%).

Experiment 1: feeding period

Experimental design, animals, and diets

A total of 72 mixed-sex piglets [(Landrace × Yorkshire) × Duroc, 36 castrated males and 36 females] with initial body weight (BW) of 7.53 ± 0.1 kg (28-day-old) were used in the experimental period of 14 weeks at Chungbuk National University research farm. They were reared on a commercially available granulated feed obtained from HANA B&F feed company (Yesan-gun, South Korea; Table 1). There were two dietary treatments: (1) Basal diet (CON), and (2) Basal diet + 0.1% silicate derived from quartz porphyry (SIL). Piglets were housed in a temperature and humidity-controlled room (24 ± 1°C; 65 ± 5%). They were randomly allotted to the two experimental diet groups according to their initial body weight and sex (12 replicate pens per treatment group and 3 piglets per pen). Each pen was equipped with a one-sided stainless-steel self-feeder and a nipple drinker that allowed piglets ad libitum access to feed and water. These diets met or exceeded nutritional requirements for pigs according to National Research Council (NRC 2012).

Table 1. Feed compositions of basal diet (as-fed basis).

Item	Weaner		Grower
	Phase 1	Phase 2
Ingredients, %
Corn	11.91	29.14	59.13
Extruded corn	28.05	20.83	–
Wheat	–	–	3.00
Soybean meal, 48% CP	23.71	30.61	30.48
Fish meal	8.00	5.00	–
Soy oil	3.70	4.50	4.29
Lactose	7.00	–	–
Whey	14.00	6.00	–
Monocalcium phosphate	1.30	1.40	1.47
L-Lys·HCl, 78%	0.54	0.53	0.34
DL-Met, 50%	0.12	0.28	0.08
L-Thr, 89%	0.22	0.21	0.03
Vitamin premix^1	0.10	0.10	0.10
Mineral premix^2	0.20	0.20	0.20
Limestone	0.95	0.90	0.63
Salt	0.20	0.30	0.25
Calculated composition
ME, kcal/kg	3,650	3,600	3,400
CP, %	21.51	20.62	17.50
Total Lys, %	1.41	1.33	0.95
Total Met, %	0.42	0.40	0.30
SID Lys, %	1.28	1.15	0.78
SID Met, %	0.39	0.36	0.24
Ca, %	0.68	0.64	0.76
Total P, %	0.52	0.49	0.42

1Provided per kg diet: 4,000 IU vitamin A, 800 IU vitamin D3, 171 IU vitamin E, 2 mg vitamin K, 4 mg vitamin B2, 1 mg vitamin B6, 16 µg vitamin B12, 11 mg pantothenic acid, 20 mg niacin and 0.02 mg biotin.

2Provided per kg diet: 220 mg Cu, 175 mg Fe, 191 mg Zn, 89 mg Mn, 0.3 mg I, 0.5 mg Co and 0.4 mg Se.

Sampling and measurements

Growth performance

Body weight (BW) of piglets were measured at the initial, middle (6 wk), and final (14 wk) experimental period. Feed consumption was recorded on a pen basis during the experiment to determine average daily gain (ADG), average daily feed intake (ADFI), and gain: feed ratio (G: F).

Nutrient digestibility

Apparent total tract digestibility (ATTD) of dry matter (DM) and nitrogen (N) were determined using 0.2% of chromic oxide as an inert indicator (Fenton and Fenton 1979). Crude protein (CP) levels were measured based on the amount of nitrogen. Pigs were fed diets mixed with chromic oxide at 6 and 14 weeks. Fresh fecal grab samples were collected from each pen and stored in a freezer at −20°C until analysed. All feed and fecal samples were analysed for DM and N following procedures outlined by the AOAC (2005). Nitrogen was determined with a Kjeltec 2300 nitrogen analyser (Foss Tecator AB, Hoeganaes Sweden). Chromium was analysed via UV absorption spectrophotometry (Shimadzu UV-1201, Shimadzu, Kyoto, Japan) following the method described by Williams et al. (1962). ATTDs of DM and N were calculated with indirect ratio methods using the following formula: coefficient of apparent total tract digestibility = (1–[(Nf × Cd)/(Nd × Cf)]) × 100, where Nf = nutrient concentration in feces (% DM), Nd = nutrient concentration in diet (% DM), Cf = chromium concentration in feces (% DM), and Cd = chromium concentration in diet (% DM).

Odour gas emission

Mixture of feces, urine and sawdust were collected from each treatment at 6 weeks and 14 weeks. 200 g mixture were stored in a plastic box (Size: 4.2 L) at room temperature (28°C) and allowed to ferment from 5 days. The plastic boxes were punctured with a roasting drill and headspace air was sampled approximately 2.0 cm above the mixture at a rate of 100 mL/min. NH₃, H₂S and VFAs concentration were measured using a gas detector tube of Gastec Corp (scope of 1.0–30.0 ppm; No. 3L for NH₃, scope of 2.5–60.0 ppm; No. 4LL for H₂S and scope of 0.25–10.0 ppm; No. 81L for VFAs).

Organ weight

At the end of experimental period, 12 piglets per treatment (1 piglet per pen) were randomly selected, weighted individually, and then killed by an intracardiac administration of sodium thiopental (Nesdonal, Merial, France, 30 mg/kg BW). Liver and spleen were removed and weighted. Organ weight was expressed as a percentage of body weight.

Experiment 2: challenge period

Experimental design

When piglets in experiment 1 reached an average BW of 28.12 ± 0.79 kg, 24 piglets in the groups of basal diet and basal diet with 0.1% silicate supplementation were randomly selected to conduct LPS challenge experiment (12 pigs per treatment and 2 pigs per pen). They were injected with Escherichia coli (Type O127: B8) lipopolysaccharide (LPS, Sigma Chemical Co, St. Louis, MO, U.S.A.) at a dose of 100 μg/kg of body weight (BW). The other half of piglets were injected with sterile saline solution at a concentration of 100 μg/kg of BW. Treatments were arranged with a 2 × 2 factorial design. Main effects were silicate and LPS challenge.

Sampling and measurements

Blood samples were collected into both non-heparinized tubes and vacuum tubes containing K₃EDTA (Becton Dickinson Vacutainer systems, Franklin Lake, NJ, U.S.A.) through jugular venipuncture at 0, 3, 6, 12, and 24 h after injection of LPS. They were collected to obtain serum and whole blood. Red blood cells (RBC), lymphocytes, and white blood cells (WBC) including neutrophil, monocyte, eosinophil and basophil counts were determined using an automatic blood analyser (ADVIA120; Bayer, Tarrytown, NY, U.S.A.). Whole blood sample was subsequently centrifuged at 3,000 × g for 15 min at 4°C and serum was harvested. Samples were then frozen and stored at –20°C until further analysis. Serum cortisol level was determined with using an automatic blood analyser (ADVIA 120, Bayer, New York, NY, U.S.A.).

Fecal microbiota

Fresh feces were obtained from 2 piglets per pen at 12 h post challenge through rectal massaging. Then 1 g of fecal sample from each pen was diluted with 9 mL of 1% peptone broth (Becton, Dickinson, Franklin Lakes, NJ, U.S.A.) and then homogenized. The count of bacteria was then conducted by plating serial 6-fold, 5-fold, and 2-fold dilutions for each onto MacConkey agar plates (Difco Laboratories, MD, U.S.A.), Lactobacilli MRS agar plates (Difco Laboratories, MD, U.S.A.), and Salmonella-Shigella agar plates to isolate Escherichia coli, Lactobacillus, and Salmonella, respectively. Lactobacillus and Salmonella were cultured at 37°C for 24 h and Escherichia coli was cultured at 37°C for 48 h.

Diarrhea score and rectal temperature

For evaluating diarrhea, feces from all pigs were scored during 24 h by checking the moisture content according to the method of Hart and Dobb (1988). A score of 3 indicated normal. A score of 2 indicated liquid feces while a score of 1 indicated watery and frothy diarrhea. Rectal temperature was determined at 0, 3, 6, 12, and 24 h post challenge using a digital electronic thermometer (WT-1, Elitech technology inc, Korea).

Statistical analysis

All data were analysed using SPSS 22.0 statistical software package (SPSS, U.S.A.). In experiment 1, t-test was used to separate treatments means. In experiment 2, all data were statistically analysed by two-way ANOVA in a 2 × 2 factorial arrangement with LPS challenge and silicate supplementation as the main effects and their interaction, using identical software. Turkey’s multiple range test was used to compare means of treatments. The level of significance was established at P < 0.05.

Results

Experiment 1

Growth performance

Pigs fed SIL diets had higher (P < 0.05) BW in 6 and 14 weeks than pigs fed CON diets (Table 2). When pigs fed SIL diet, ADG and G: F ratio was increased (P < 0.05) compared to that of pigs fed CON diets in experimental period. However, there was no significant (P > 0.05) difference in ADFI between the two treatments in all experimental period.

Table 2. Effects of silicate supplementation on growth performance in weaning to growing pigs (Exp. 1)^1.

Items^2	CON	SIL	SE^3	P-value
Body weight (kg)
Initial	7.53	7.53
6 wk	27.34	28.95	0.89	0.04
14 wk	75.39	79.23	1.97	0.05
ADG (g/d)
0 – 6 wk	472	510	9.38	0.05
6 – 14 wk	858	898	14.46	0.04
0 – 14 wk	692	732	7.09	0.04
ADFI (g/d)
0 – 6 wk	833	814	21.54	0.95
6 – 14 wk	2167	2135	87.01	0.58
0 – 14 wk	2085	2054	21.98	0.67
G: F (g/g)
0 – 6 wk	0.52	0.62	0.05	0.04
6 – 14 wk	0.40	0.51	0.01	0.03
0 – 14 wk	0.34	0.37	0.02	0.04

1CON, basal diets based; SIL, supplementation of 0.1% silicate in basal diets.

2ADG, average daily gain; ADFI, average daily feed intake; G: F, feed efficiency.

3Pooled standard error.

^a,bMeans in the same row with different superscripts differ (P<0.05).

Nutrient digestibility

Pigs fed SIL diets had higher (P < 0.05) DM and CP digestibility than pigs fed CON diets at 6 weeks (Table 3). Supplementation with silicate did not significantly (P > 0.05) affect DM digestibility although it increased (P < 0.05) CP digestibility compared to the CON diet at 14 weeks.

Table 3. Effects of silicate supplementation on nutrient digestibility in weaning to growing pigs (Exp. 1)¹.

Items (%)	CON	SIL	SE^2	P-value
6 wk
Dry matter	73.63	75.33	1.28	0.04
Crude protein	69.12	73.33	1.96	0.03
14 wk
Dry matter	74.25	75.02	1.22	0.67
Crude protein	65.45	69.32	1.05	0.03

¹CON, basal diets based; SIL, supplementation of 0.1% silicate in basal diets.

2Pooled standard error.

^a,bMeans in the same row with different superscripts differ (P < 0.05).

Odour gas emission

Supplementation with silicate decreased (P < 0.05) NH₃ emission compared with CON group while H₂S and VFAs emission did not be affected (P > 0.05) in 6 weeks (Table 4). Also, NH₃ emission decreased (P < 0.05) while H₂S and VFAs did not affected (P > 0.05) when pigs were fed SIL diet at 14 weeks.

Table 4. Effects of silicate supplementation on odour gas emission in weaning to growing pigs (Exp. 1)¹.

Items (ppm)	CON	SIL	SE^2	P-value
6 wk
H2S	3.23	2.93	0.35	0.08
NH3	15.14	10.27	1.89	0.02
VFAs	1.34	1.37	0.09	0.86
14 wk
H2S	4.24	4.01	1.23	0.49
NH3	18.24	15.32	1.12	0.04
VFAs	1.62	1.75	0.15	0.57

¹CON, basal diets based; SIL, supplementation of 0.1% silicate in basal diets.

²Pooled standard error.

^a,bMeans in the same row with different superscripts differ (P < 0.05).

Organ weight

There was no significant (P > 0.05) difference in organ weight related to immune traits between the two treatment groups (Table 5).

Table 5. Effects of silicate supplementation on organ weight in weaning pigs (Exp. 1)¹.

Items (%)	CON	SIL	SE^2	P-value
Liver	3.47	3.74	0.32	0.56
Spleen	0.26	0.34	0.21	0.21

1CON, basal diets based; SIL, supplementation of 0.1% silicate in basal diets.

2Pooled standard error.

^a,bMeans in the same row with different superscripts differ (P < 0.05).

Experiment 2

Immune traits

There were significant (P < 0.05) interactive effects between diets and LPS on RBC content at 6 h post challenge (Table 6). Piglets fed SIL diet had higher (P < 0.01) WBC content than piglets fed CON diet at 24 h post LPS challenge. WBC content decreased (P < 0.05) at 3–24 h post challenge with LPS. There were also significant (P < 0.05) interactive effects between diets and LPS on WBC at 24 h post challenge. At 3–24 h after challenge, piglets fed SIL diets had higher contents of neutrophils (P < 0.05) than piglet fed CON diets. Contents of neutrophils were increased (P < 0.05) when piglets were injected with LPS. There were also significant (P < 0.05) interactive effects between diets and LPS. Monocyte content was increased (P < 0.05) when piglets were fed silicate. At 3, 6, and 12 h post LPS challenge, eosinophil content was increased (P < 0.05). At 6 and 12 h post LPS challenge, basophil content was increased. LPS challenge increased (P < 0.05) lymphocyte content at 3–24 h after post challenge compared to LPS non-challenged control. At 3 and 24 h post LPS challenge, the pigs fed with silicate and challenged LPS had higher (P < 0.05) lymphocyte content than the pigs fed with CON diet and non-challenged LPS. There were also significant (P < 0.05) interactive effects between diets and LPS on lymphocyte content at 24 h post LPS challenge. Cortisol content was increased in piglets at 3, 12, and 24 h after LPS challenge. At 12 and 24 h post LPS challenge, the pigs supplemented with silicate had lower (P < 0.05) cortisol content than the pigs fed with CON diet. There were also significant (P < 0.05) interactive effects between diets and LPS on lymphocyte content at 24 h after LPS challenge.

Table 6. Effects of silicate supplementation on immune traits in weaning pigs challenged by lipopolysaccharide (Exp. 2)^1.

Items^2	CON		SIL		SE^3	P-value^4
	LPS -	LPS +	LPS -	LPS +		D	L	D×L
RBC (10^6/㎕)
0h	6.94	6.57	6.91	7.44	0.24	0.13	0.74	0.09
3h	6.68	6.51	7.07	6.88	0.17	0.33	0.15	0.05
6h	6.90	6.57	6.80	7.46	0.21	0.12	0.45	0.03
12h	6.73	6.57	6.70	6.17	0.23	0.39	0.18	0.45
24h	6.98	6.08	7.03	6.71	0.67	0.36	0.20	0.99
WBC (10^3/㎕)
0h	24.21	20.54	25.93	27.03	2.54	0.13	0.62	0.37
3h	23.75	7.25	28.30	8.20	1.68	0.13	0.01	0.09
6h	27.01	13.97	30.95	17.07	2.33	0.16	0.01	0.86
12h	23.86	16.48	30.30	20.97	2.93	0.09	0.02	0.75
24h	21.00	19.12	25.17	30.79	2.38	0.01	0.05	0.02
Neutrophil (%)
0h	23.60	30.95	28.58	33.20	2.13	0.42	0.19	0.76
3h	36.28	65.13	38.43	57.53	4.11	0.08	0.01	0.43
6h	44.15	58.10	37.97	46.85	3.13	0.02	0.01	0.43
12h	41.83	53.80	35.95	44.58	5.29	0.18	0.08	0.76
24h	40.55	46.40	35.09	42.21	1.78	0.02	0.03	0.73
Monocyte (%)
0h	3.88	2.83	2.10	3.63	1.56	0.76	0.88	0.43
3h	3.00	1.90	2.43	3.08	0.45	0.52	0.63	0.08
6h	3.58	3.10	2.53	2.50	0.59	0.19	0.68	0.71
12h	5.35	4.75	3.55	4.80	1.30	0.52	0.81	0.49
24h	1.00	2.00	2.75	1.80	0.12	0.01	0.84	0.01
Eosinophil (%)
0h	1.30	1.88	1.30	1.55	0.32	0.62	0.23	0.62
3h	0.37	1.55	1.03	1.80	0.41	0.30	0.04	0.63
6h	1.00	1.50	0.50	1.73	0.29	0.65	0.01	0.23
12h	0.48	1.40	0.13	1.43	0.21	0.45	0.01	0.38
24h	1.75	1.75	1.70	2.10	0.26	0.29	0.89	0.18
Basophil (%)
0h	1.05	0.85	0.83	1.53	0.21	0.29	0.25	0.12
3h	0.97	0.75	1.03	1.18	0.30	0.44	0.91	0.56
6h	0.73	1.13	0.80	1.33	0.14	0.31	0.01	0.61
12h	0.45	0.63	0.27	0.73	0.12	0.76	0.03	0.28
24h	0.70	0.70	0.70	0.75	0.05	0.12	0.23	0.23
Lymphocyte (%)
0h	33.88	37.80	32.05	37.25	5.10	0.54	0.30	0.94
3h	31.63	58.43	30.35	55.93	4.04	0.01	0.01	0.33
6h	30.43	57.65	32.73	46.30	3.06	0.10	0.01	0.04
12h	35.53	56.30	35.90	55.85	5.70	0.32	0.01	0.45
24h	29.15	56.30	24.85	45.85	3.82	0.01	0.01	0.01
Cortisol (ug/dL)
0h	2.07	2.12	1.49	2.21	0.64	0.71	0.56	0.61
3h	3.27	5.68	1.71	6.72	0.65	0.70	0.01	0.09
6h	3.82	5.24	2.85	3.76	0.88	0.42	0.08	0.41
12h	2.87	5.19	1.82	3.03	0.21	0.01	0.01	0.02
24h	2.66	4.33	2.32	2.26	0.14	0.01	0.01	0.01

¹CON, basal diets based; SIL, supplementation of 0.1% silicate in basal diets; LPS -, without lipopolysaccharide injection; LPS +, with lipopolysaccharide injection at 0.01%.

²RBC, red blood cell; WBC, white blood cell.

³Pooled standard error.

⁴D, diet; L, LPS; D×L, interaction between diet and LPS.

Fecal microbiota

Lactobacillus or Salmonella count was not significantly (P > 0.05) affected by diet, LPS, or their interaction (Table 7). Piglets fed SIL diet had lower (P = 0.05) Escherichia coli counts than piglets fed CON diet. LPS challenge significantly (P < 0.05) increased Escherichia coli counts in feces.

Table 7. Effects of silicate supplementation on fecal microbiota in weaning pigs challenged by lipopolysaccharide (Exp. 2)¹.

Items (log10cfu/g)	CON		SIL		SE^2	P-value^3
	LPS -	LPS +	LPS -	LPS +		D	L	D×L
Lactobacillus	8.65	8.67	8.77	8.72	0.25	0.52	0.90	0.77
Escherichia coli	7.59	7.98	7.49	7.84	0.05	0.07	0.02	0.21
Salmonella	2.96	3.00	2.99	2.98	0.13	0.97	0.90	0.85

¹CON, basal diets based; SIL, supplementation of 0.1% silicate in basal diets; LPS -, without lipopolysaccharide injection; LPS +, with lipopolysaccharide injection at 0.01%.

²Pooled standard error.

³D, diet; L, LPS; D×L, interaction between diet and LPS.

Diarrhea score and rectal temperature

Based on diarrhea score, supplementation with silicate decreased (P < 0.05) diarrhea incidence compared to CON diet at 3 and 24 h after LPS challenge (Table 8). At 3–24 h, piglets challenged with LPS had higher (P < 0.05) diarrhea incidence than piglets not challenged with LPS. There were significant (P < 0.05) interactive effects between diets and LPS on diarrhea incidence at 24 h post LPS challenge. Piglets challenged with LPS had higher (P < 0.05) rectal temperature than piglets not challenged with LPS at 3, 6, and 24 h (Table 9). At 6 and 24 h post LPS challenge, supplementation of silicate decreased (P < 0.05) rectal temperature. There were significant (P < 0.05) interactive effects between diets and LPS on rectal temperature at 6 and 24 h post LPS challenge.

Table 8. Effects of silicate supplementation on diarrhea score in weaning pigs challenged by lipopolysaccharide (Exp. 2)¹.

Items	CON		SIL		SE^2	P-value^3
	LPS -	LPS +	LPS -	LPS +		D	L	D×L
0h	1.50	2.50	2.75	2.75	0.20	0.22	1.00	1.00
3h	2.13	1.00	2.50	1.50	0.19	0.03	0.01	0.76
6h	2.50	1.25	2.62	1.63	0.23	0.23	0.01	0.54
12h	2.50	1.50	2.88	1.75	0.22	0.16	0.01	0.77
24h	2.50	1.75	2.87	2.75	0.21	0.01	0.04	0.04

¹CON, basal diets based; SIL, supplementation of 0.1% silicate in basal diets; LPS -, without lipopolysaccharide injection; LPS +, with lipopolysaccharide injection at 0.01%.

²Pooled standard error.

³D, diet; L, LPS; D×L, interaction between diet and LPS.

Table 9. Effects of silicate supplementation on rectal temperature in weaning pigs challenged by lipopolysaccharide (Exp. 2)¹.

Items (°C)	CON		SIL		SE^2	P-value^3
	LPS -	LPS +	LPS -	LPS +		D	L	D×L
0h	39.43	39.38	39.25	39.58	0.16	0.94	0.42	0.28
3h	40.25	42.95	40.30	42.70	0.17	0.98	0.01	0.78
6h	40.18	41.98	40.33	40.75	0.11	0.01	0.01	0.01
12h	40.27	40.88	40.33	40.33	0.16	0.14	0.09	0.09
24h	39.75	40.40	39.40	39.35	0.13	0.01	0.05	0.02

¹CON, basal diets based; SIL, supplementation of 0.1% silicate in basal diets; LPS -, without lipopolysaccharide injection; LPS +, with lipopolysaccharide injection at 0.01%.

²Pooled standard error.

³D, diet; L, LPS; D×L, interaction between diet and LPS.

Discussion

Experiment 1

Growth performance

Results of this experiment showed that supplementation with silicate improved BW, ADG, and G: F ratio compared with control diet in the 6 and 14 weeks. This result agreed with previous results showing that addition of silicate to diets has positive effects on growth performance and feed efficiency (Trckova et al. 2009; Yan et al. 2011; Duan et al. 2013). However, other studies have reported that the addition of silicate does not affect the growth performance of weaning pigs (Poulsen and Oksbjerg 1995; Xia et al. 2005). Song et al. (2012) have reported no significant effect of silicate on growth performance when different silicates (smectite, kaolinite, and zeolite) are added to diets at 0.3% alone or in combination. These differences might be due to differences in types and dosages of silicate as well as genotypes of pigs used in the experiment.

Nutrient digestibility

In our study, the DM and CP digestibility in pigs fed SIL diet were increased, which agrees with the results of Parisini et al. (1999) who reported that CP and energy digestibility are improved in the group fed with diet added with 2% silicate mineral (sepiolite) by 6.1% and 5.3%, respectively, compared to those in the control group. Also, Yan et al. (2011) showed that the addition of silicate significantly improved DM and N digestibility at 7 days without affecting them at 21 days. Many researchers reported that these improvements of nutrient digestibility were because silicate caused the reduction of feed movement in intestine and the secretion of more digestive enzyme (Xia et al. 2004; Trckova et al. 2009; Vondruskova et al. 2010; Safaeikatouli et al. 2012; Lv et al. 2015). The improvement in growth performance when weaning piglets fed with diet containing 0.1% silicate might be also due to improved nutrient digestibility which increased their growth rate.

Odour gas emission

Addition of 0.1% silicate did not affect odour causing compound like H₂S and VFAs, while NH₃ emission reduced compared with pigs fed CON diets in 6 and 14 weeks. The results in this experiment is in partial agreement with the report by Yan et al. (2010, 2011) who proved that 0.3% and 0.6% clay mineral decreased H₂S and NH₃ levels compared with pigs were fed reduced when pigs were fed basal diets, respectively. Therefore, reduction of odour gas emission in this experiment is considered to be due to ability of silicate such as ion exchange capacity and improvement of CP digestibility.

Organ weight

There was no significant difference in the weight of liver or spleen as immune organ between the two treatment groups. Because liver and spleen have Kuffer cells and splenic macrophage, respectively, these organs are responsible for the defense in the body (Cho et al. 1990). The spleen also stores half of mononuclear cells to heal wounds. Currently there is no research showing the relationship between silicate as antibiotic alternative and immune organ in pig. Thus, this study hypothesized that relative organ weight-related immune traits might be stimulated by silicate to show increase in size. In this experiment, there was no significant difference in organ weight between the two treatment groups. Although 0.1% addition of silicate did not affect immune traits, further research is needed to find the appropriate amount of additive that can have a positive effect on the immune system.

Experiment 2

Immune traits

Wright et al. (2000) have reported that the inflammatory response by LPS can last from 12 to 24 h. In this study, RBC did not affect by SIL and LPS, while WBC was increased compared to that in the group without LPS challenge at 3–24 h after LPS challenge. However, previous studies have reported that piglets injected with LPS have lower WBC than piglets not injected with LPS (Chen et al. 2009; Guo et al. 2017). This difference is considered that silicate eluted from quartz porphyry might effectively play a role in preventing death and denaturation of blood cells and moderately maintaining cells during the inflammatory response. Neutrophils are white blood cells that have the largest percentage (40–75%) in most mammals. They are one of first cells in response to a specific situation such as bacterial infection, exposure to environmental stimuli, and certain cancers (Waugh and Wilson 2008; Jacobs et al. 2010). 0.1% silicate supplementation increased neutrophils compared to CON diets at 6–24 h post LPS challenge. Thus, it could be considered that silicate used in this study could improve immunity against infection and disease. Monocytes as a subset of WBCs can be significantly increased by hazard substance such as mycotoxin and deoxynivalenol (Pinton et al. 2008; Weaver et al. 2013). However, there was no significant difference in monocytes after LPS challenge. These differences might be because previous studies used fungi directly while the present study used LPS as one of cell wall components of Gram-negative bacteria. Also, piglets fed diet containing 0.1% silicate had higher monocytes than piglets fed CON diet at 24 h post LPS challenge. However, the results of monocyte did not have a constant tendency, so it is considered that additional studies are needed. Eosinophils account for 1–3% of white blood cells. They are increased in white blood cells during allergies and parasites infection (Dombrowicz and Capron 2001). Also, Basophil is one of white blood cell that is responsible for restricted phagocytosis such as neutrophils and monocytes in the blood. 0.1% silicate supplementation did not significantly affect eosinophils and basophil content while LPS challenge treatments increased compared with non-LPS challenge treatments. These increases might be due to immune response by LPS. Lymphocytes are blood cells that account for 30% of white blood cells. They have the longest survival period. Piglets injected with LPS had higher levels of lymphocytes at 3–24 h post challenge than piglets without LPS challenge. Also, 0.1% silicate increased levels of lymphocytes compared to CON diet. In the present study, proliferations of white blood cells such as neutrophils, monocytes, and lymphocytes were enhanced when piglets were fed diets supplemented with silicate. This result agreed with results of Yuan et al. (2004) and Li and Kim (2013). They reported that lymphocytes and monocytes were increased when piglets were fed diets with 0.5% biotite or 0.5–1.0% sericite. In addition, there were significant interactions between diets and LPS challenge. This meant that piglets in the group fed with 0.1% silicate had lower lymphocyte counts after injection with LPS than piglets fed with CON diets and injected with LPS. These observations agreed well with results of Russell et al. (2016). They reported that immunoglobulin G (IgG) and immunoglobulin A (IgA) levels were increased when female mice fed with 50 µg nanoscale silicon dioxide were challenged by H1N1 influenza virus compared with mice treated by antigen alone. Cortisol level increased by LPS challenge, while 0.1% silicate supplementation decreased. Also, there were significant interaction effects between diet and LPS, which meant that 0.1% silicate alleviated piglets’ stress, thereby they enhanced immune capacity of piglets.

Fecal microbiota

Lactobacillus or Salmonella count was not affected by diet, LPS, or their interaction (Table 6). However, Escherichia coli count tended to be decreased when piglets were fed 0.1% silicate. LPS challenge increased Escherichia coli count in feces. These results were due to improvements of protein digestibility. However, Thacker (2003) has reported that increasing level of silicate in diets does not affect the number of Lactobacillus, Enterobacteria, or Salmonellas. These conflicting results might be due to differences in types and dosages of silicate used in the experiment. Additionally, we inferred that the reduction of Escherichia coli was due to ion exchange capacity of silicate and improvement of protein digestibility (Wellock et al. 2008; Song et al. 2012).

Diarrhea score and rectal temperature

Diarrhea caused by Escherichia coli is a common disease in weaning pigs. It causes economic losses of farms by increasing death rates and decreasing growth performance (Fairbrother et al. 2005). Also, it causes poor intestinal health, resulting in reduced nutrient absorption and increased infection rate. Many studies have reported that silicate has a positive effect in improving diarrhea in pigs (Schell et al. 1993; Trckova et al. 2009; Song et al. 2012). In the present study, LPS challenge increased the incidence of diarrhea at 3–24 h. Addition of 0.1% silicate significantly alleviated the incidence of diarrhea compared to CON diets at 3 and 24 h after LPS challenge. This result agreed with results of previous studies showing that silicate reduced the incidence, severity, and duration of diarrhea (Papaioannou et al. 2004; Xia et al. 2004; Almeida et al. 2013). Therefore, it was considered that silicate derived from quartz porphyry could alleviate the incidence and severity of diarrhea by reducing the number of Escherichia coli in the intestine. Many LPS related studies have shown that rectal temperature of pigs is increased after LPS challenge (Turner et al. 2001; Yi et al. 2005; Wang et al. 2011; Kim et al. 2012). Results of the present study also agreed with results of prior studies. At 3, 6, and 24 h after post LPS challenge, piglets injected with LPS had higher rectal temperature than piglets without LPS challenge (Table 8). Addition of 0.1% silicate reduced rectal temperature at 6 and 24 h post LPS challenge. There was also an interaction between diets and LPS which significantly improved rectal temperatures of pigs fed with 0.1% silicate and challenged with LPS. As a result, silicate eluted from quartz porphyry as antibiotic alternative had a positive effect on the immune system.

Conclusion

In conclusion, Addition of 0.1% silicate to diets improved growth performance and nutrient digestibility of weaning piglets non-challenged LPS. In addition, immune capacity, fecal microbiota, and incidence of diarrhea were improved when pigs were fed diets supplemented with 0.1% silicate and challenged LPS.

Disclosure statement

No potential conflict of interest was reported by the author(s).