Effects of inorganic minerals replaced by complexed glycinates on growth, blood profiles, immune responses, intestinal morphology, and mineral excretion in piglets


 BACKGROUND: The effects of inorganic trace minerals (ITM) replaced by low-dose glycine-complexed trace minerals (GCM) on growth, serum parameters, immunity, intestinal morphology, and mineral excretion in piglets were investigated. One hundred and twenty-eight weaned piglets (14.18 ± 0.33kg body weight (BW)) were randomly assigned to 4 treatments with 4 replicates, 8 piglets per replicate. Treatments consist of: (T1) basal diet + 100% inorganic trace mineral (ITM) as the control group (20 ppm Cu, 150 ppm Fe, 150 ppm Zn, and 30 ppm Mn from sulfates); (T2) basal diet + 50% ITM (Cu, Fe, Zn, and Mn from sulfates, 50% of control) + 50% organic trace minerals (OTM, Cu, Fe, Zn, and Mn from glycine complexed trace minerals (GCM), 50% of control); (T3) basis diet + 50% OTM from GCM; (T4) basal diet + 70% OTM from GCM. The feeding period lasted 28 d and was divided into 2 stages (0 to 14 d and 15 to 28 d). After feeding trial , 6 pigs per treatment were randomly selected to slaughter for sampling. RESULTS: Average daily gain, feed intake, and G:F were not affected by dietary treatments during the overall period. During the second, and the overall feeding phases, the digestibility of Zn and Fe in T3 and T4 was higher than that of T1 ( P < 0.05). The concentration of serum ferritin in T2 was significantly higher than T3 and T4. Serum immunoglobulin A concentration in the ileal mucosa of T2 was higher than that of T1 ( P < 0.05), and the higher duodenum villus height was observed in T4 compared with the rest treatments ( P < 0.05). The lowest trace mineral excretion was overserved in T3 ( P < 0.01); in addition, the urinary concentrations of Zn and Fe in T2 were lower than that in T1 ( P < 0.05). CONCLUSION: These results indicate that GCM have higher bioavailability than ITM, and that supplementation of low-dose GCM to replace full dose ITM could reduce mineral excretion without affecting performance, blood profiles, immune responses, and intestinal morphology in piglets.


Introduction
parameters, immunity, intestinal morphology, and fecal mineral excretion in piglets.

Experiment Design and Animal Management 76
The site and piglets of the feeding trial were provided by Anji Zhengxin Breeding Farm (Huzhou, China). A 77 total of 128 pigs with an initial BW of 14.18 kg (SEM = 0.33) were allotted to 4 dietary treatments in a 78 randomized complete block design with 4 replicates (pens) and 8 animals per pen. All piglets were 35 days old 79 and weaned at 23 days of age. Pigs were acclimatized for 5 days. The formal feeding trial started when pigs were 80 40 days old and lasted for 28 days, which was divided into two phases. Phase Ⅰ was 0 to 14 d and phase Ⅱ was 14 81 to 28 d. The basal diet (Table 1)    Pens used in the feeding trial had hard concrete slotted flooring and were equipped with nipple drinkers and 94 stainless-steel feeders. All piglets were allowed ad libitum access to feed and water. Room temperature and 95 ventilation were controlled automatically according to the standard procedures of the farm. Pigs were weighed 96 on at day 1, 15, and 28 of the trial and the feed intake was also recorded at the last day of each phase. Upon 97 finishing the feeding trial, 6 pigs per treatment (at least one pig per pen) were randomly selected for slaughtering 98 and sampling. Average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F) were 99 calculated at the end of the trial.

Sample Collecting 101
Fresh feces collection was carried out on the last three days of each phase on the pen basis, mixed and stored feces sample of each phase at -80 ℃ for later analysis. Before slaughtering, blood samples were collected 103 by syringe from the precaval vein of selected pigs. Serum was obtained by centrifuging at 1260×g at 4 ℃ for 10 7 syringe and stored at -80 °C. The small intestine was separated and dissected. The segments of middle duodenum 106 and jejunum were separated for a length of 2 cm with flushing by sterile saline solution and stored in 4% 107 paraformaldehyde under 4 °C. Additional jejunum segment was isolated and stored in 2.5% malondialdehyde 108 under 4 ℃. Duodenum, jejunum and ileum mucosa were scraped and collected using the clean glass slides and 109 stored at -80 °C.

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Concentrations of serum inflammatory factors IL-6, IL-10, and TNF-α were determined by ELISA kits (Beijing 119 4A Biotech Co., Ltd, Beijing, China). The jejunum and ileum mucosa were ground on ice by a glass grinder to 120 make a 10% homogenate for the detection of sIgA concentration, and the procedure was also carried out 121 according to the instruction of commercial kit (Jiancheng Bioengineering Institute, Nanjing, China).

Mineral Measurement and ATTD Calculation 123
The trace mineral concentrations in feed, feces and urine samples were determined by atomic flame 124 absorption spectrometry (ICE-3500, Thermo Crop., USA). Before the analysis, feces and feed samples were 125 dried at 65 ℃ for 48 h, and then pulverized by a grinder and passed through a 60 mesh sieve (0.25 mm) to 126 achieve homogenous samples. The ground feces and feed sample were digested using the method described as 127 follows: 1 g of sample was placed in a crucible and ashed at 550 ℃ for 4 h in muffle furnace until it became 128 off-white powder, and 10 ml of 10% hydrochloric acid was added for dissolving. After filtration, the filtrate was 129 diluted to 100 ml with distilled water. Urine sample was thawed and treated by microwave wet digestion as 130 follows: 1 ml sample was mixed with 6 ml nitric acid for 3 minutes at 120 ℃ in a microwave digestion system,

Intestinal Morphology Analysis and Scanning Electron Microscopy (SEM) 138
After adequate fixation, small intestine samples were embedded in paraffin, cut cross-section of 5 μm and 139 then stained with hematoxylin and eosin (H&E, Solarbio Science & Technology Co., Ltd, Beijing, China).

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Images were acquired using a DM3000 microscope (Leica, Wetzlar, Germany). Villous height (VH) and crypt 141 depth (CD) were measured using Image-Pro software (Media Cybernetics, MD, USA) as previously reported 142 [8,9]. All images and measurements were performed by the same person using the same microscope. The 143 jejunum segment in glutaraldehyde was cut into a small rectangle before osmic acid fixation. Trimmed jejunum 144 samples were rinsed three times with PBS and then fixed with 1% OsO4 for 1.5 h, then osmic acid was discarded 145 and dehydrated in a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 20 min at each

Metal Transporter mRNA Expression in Duodenum 151
An analysis of duodenum metal transporter gene expression was conducted as previously described by

Growth Performance 174
The average daily gain in each phase was similar among the treatments (Table 4). In phase 1, ADFI of T2 175 was significantly higher than that of T4. In phase 2, the G:F of piglets in 100% inorganic trace minerals 176 treatment and the mixture of 50% glycine complexed trace minerals with 50% inorganic trace minerals treatment 177 (T1 and T2) were significantly higher than that of piglets in low-dose groups (T3 and T4).

Apparent Total Tract Digestibility of Trace Minerals 185
As shown in Table 5, no differences in ATTD of tested minerals were detected in the first period (0 to 14 d); 186 in the second period and the overall feeding period, the ATTD of zinc and iron in the low-dose GCM treatments 187 (T3 and T4) were higher than that in the 100% inorganic treatment (T1). 198 Table 6 shows the results of serum biochemical indicators tested. The concentration of serum ferritin in T2 199 was significantly higher than that of T3 and T4 and numerically higher than T1 as well; however, supplemental 200 glycine complexed trace minerals to replace inorganic trace minerals did not affect other serum parameters 201 tested.

Immune Response 203
There were no significant differences on the concentration of serum IgA, IgG, IgM, C3 and C4 among the 13 four treatments (Fig. 1A). The similar results were also found in serum inflammatory factors (TNF-α, IL-6 and 205 IL-10) as shown in Fig. 1B. However, the sIgA concentration of ileum in T2 was significantly higher than that of 206 T1 (Fig. 1C).

Intestinal Morphology 208
The duodenal VH in 70% GCM treatment was significantly higher than that in other 3 treatments; similarly,

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the v/c of T4 was also numerically higher than the other three treatments (Fig. 2D). In the jejunal morphology, 210 VH and CD of T4 was higher than that of T1, and T3 had the highest v/c though it did not reach the significant 211 difference (Fig. 2E). The SEM images of the jejunum showed that intestinal villi from T3 and T4 were more 212 intact than that of T1 and T2 (Fig. 2C). Under 20,000 times magnification, the jejunum microvilli of T3 and T4 213 were more serried than T1 and T2 (Fig. 2C).

Relative Expression of Metal Transporter mRNA 215
In Fig. 3, the relative expression of ZnT1 mRNA in T4 was higher (P < 0.05) than that in the other three  Table 7 shows that the amount of trace minerals excreted in urine and feces were positively correlated with 220 the dietary supplemental mineral level. The trace minerals excretion in urine and feces of low-dose treatments 221 (T3 and T4) were lower (P < 0.05) than the commercial level minerals treatments (T1 and T2), and the trace 222 minerals excretion in T3 was also lower (P < 0.05) than T4. When 50% of dietary ITM replaced by OTM from 223 Glycinates, the excretion amount of Zn and Fe in urinary sample of T2 was lower (P < 0.05) than that of T1.

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The trace minerals required for growth and development of piglets after weaning are mainly from innate 232 reserves and diets. Due to the incomplete development of intestinal and low feed intake within a period of time 233 after weaning, the trace minerals taken by piglets from the diet are limited. Therefore, providing the piglets with 234 higher bio-available trace minerals may be an effective method to reduce the amount of trace minerals used. In 235 our study, we evaluated the effects of low-dose glycine-complexed trace minerals replacing ITM in piglets.

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Nowadays, livestock is generally fed diets that are formulated to provide an excess of trace minerals to 237 maximize growth performance. The growth-promoting mechanism of trace minerals as Cu and Zn is mainly that 238 a high concentration of metal ions could inhibit the growth of intestinal microbes and stimulate the release of 239 neuropeptide Y, which results in increase of feed intake, maintain the body's immune function and antioxidant T1 with commercial level of supplemental ITM, and the feed intake of the partial replacement treatment (T2) 242 was numerically higher than that of other 3treatments in phase 1. Thus, higher feed intake and Cu, Zn 243 concentration may be the reason for the greater ADG and G:F in T2 compared with other 3 treatment groups.

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However, during the overall feeding period, low doses of glycine-complexed trace minerals did not have a 245 significant impact on piglet performance, which is similar to Feng's findings [16]. The vulnerable intestinal 246 mucosa of piglets is susceptible to damage due to conversion from milk to solid feed after weaning. The pigs 247 treated with glycine-complexed trace minerals had more intact intestinal villi in that the jejunum microvilli of 248 pigs in T3 and T4 were more serried than that in T1. We speculated that this may be due to the fact that glycine

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Serum antibodies are the essential component of innate and adaptive immunity and immunological memory.

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and IgM) and complements 3 and 4 were not affected by the level and source of trace minerals, which is similar 284 to the results of Feng's research [27] in which piglets were fed with iron glycine chelate. This indicates that the minimum treatment dose in this study was not sufficient to affect humoral immunity. The weaning process of 286 piglets is often accompanied by inflammation, which can trigger up-regulation of pro-inflammatory cytokines 287 that can damage the mucosal barrier and intestinal permeability, such as TNF-α, IL-6. In Pu's study [28], 288 supplementation iron dextran to newborn piglets reduced the expression of inflammatory factors TNF-α and IL-6 289 mRNA, confirming that iron supplementation can alleviate the increase in inflammatory factors caused by iron 290 deficiency. In present study, we found that serum concentrations of pro-inflammatory factors (TNF-α, IL-6) and

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The intestinal mucosa is formed by monolayer columnar epithelium cells, performing the primary functions 302 in digesting, absorbing of nutrients, and preventing luminal pathogens and toxic substances cause any damage.

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Early weaning stress syndrome usually leads to a decrease in VH and intestinal dysfunction in piglets. Impaired

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reported that jejunum VH of weanling pigs from sows fed ZnSO4 or Zn-AA was greater than those from sows 307 optical microscopy and electron microscopy (×150) also showed that the jejunum villi morphology in T3 and T4 311 were more intact. At 20,000× magnification, the jejunum villi of T3 and T4 were also denser. This may be 312 related to the nutritional function of glycine in the GCM discussed above.

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The intact intestinal villus morphology not only reduces local inflammation and diarrhea in the intestine, 314 but also facilitates the absorption of nutrients. In the apparent total tract digestibility of trace minerals, glycine 315 complexed trace minerals exhibited higher digestibility than inorganic trace minerals, and there were significant 316 differences in iron and zinc. ZnT1, Fpn1 and Ctr1 are specific transporters of zinc, iron and copper, respectively. and Fpn1 mRNA in T4 was consistent with the aforementioned high apparent digestibility. These two results 326 demonstrate that the digestibility of glycine complexed iron and zinc is higher than that of inorganic iron and 327 zinc. In the two treatments supplemented with 100% trace minerals, there was no significant difference in ATTD 328 of trace minerals, but in the results of trace mineral emissions from urine and feces, T2 had lower trace mineral emissions than T1 (except Mn), the concentration of urine zinc and urine iron in T2 was significantly lower than 330 that in T1. Since the digestibility of glycine complexed trace minerals is higher than that of inorganic trace 331 minerals, compared with 100% ITM treatment, 50% ITM+50% GCM treatment has more metal ions absorbed 332 into the body, while the excretions were also lower. This indicates that glycine complexed trace minerals have 333 higher bioavailability and are more easily selected for storage. Although the metal excretions of T3 and T4 were 334 lower, it is difficult to conclude whether this was due to the difference in the amount of addition or

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We sincerely thank Anji Zhengxin Animal Husbandry Co.,Ltd, Zhejiang, China for providing the feeding 362 ground and weaned piglets. We would like to thank Shunxin Shen and Qiuming Shen for the support of feeding 363 and management of piglets.