Dietary ZnO and arginine supplementation on the dynamic change of microbiota, intestinal morphology, and immune function of weaned pigs subjected to heat stress CURRENT STATUS:

Background. Weaning stress is an economically important problem in the pigs, and the economic loss of the growth performance reduction is even more critical if the heat stress adds to the weaning stress. The supplementation of ZnO is an effective option in reducing the adverse effects of weaning time. This study aimed to investigate the effect of the L-arginine (Arg) inclusion and different doses of ZnO to determine the best dietary supplementation ratio on growth performance, intestinal microbiota and integrity, and immune status in weaned pigs. A total of 180 weaned pigs (28 day-old) were randomly allotted to six treatments with 6 replicate pens in each treatment and 5 pigs per pen. The dietary treatments were: control diet (Con; with 1.1% Arg and without ZnO supplementation); Con + 2500 ppm Zn as ZnO (P-Zn); Con + 1.6% Arg (ARG); Con + 500 ppm of Zn as ZnO + 1.6% Arg (ZnArg1); Con + 1000 ppm of Zn as ZnO + 1.6% Arg (ZnArg2); P-Zn + 1.6% Arg (ZnArg3). Results. The overall result showed that the inclusion of ZnArg3 significantly improved the average daily gain compared with the Con treatment. There was a reduction of feed intake in the Con diet compared with the ZnArg3 diet at phase 1 and overall. At phase 1, the weaned pigs in the ZnArg3 and P-Zn groups exhibited the decreased population of Clostridium spp. in the ileum compared with those of the Con group. In addition, a lower ileal Clostridium spp. population was detected in the ZnArg2 pigs compared with the Con pigs. At phase 2, the colonization of Clostridium spp. was higher in the Con and ARG treatments compared with ZnArg3 treatment. The weaned pigs fed the ZnArg1 and ZnArg3 diets showed a greater villus height of duodenum compared with the Con and P-Zn treatments. The count of eosinophil was significantly higher in the Con and ZnArg1 compared with the ZnArg2

weaned pigs compared with the Con group. The jejunum gene expression of TLR4 was upregulated in the Con and ARG treatments compared with the ZnArg1 and ZnArg3. The ZnArg1, ZnArg2, and ZnArg3 treatments showed a lower mRNA expression of TNF-α compared with the Con group.
Conclusion. The Arg supplementation did not improve the growth performance, microbial composition, or immune status of weaned pigs but showed a similar growth performance when supplemented with 500 ppm Zn as ZnO compared with 2500 ppm Zn as ZnO.

Introductions
The health condition of piglets is highly unstable at weaning time due to the stress of changing feed form and reduced feed intake [1][2][3]. The decrease of voluntary feed intake and being separated from mother sow increase the stress and may cause dysbiosis due to sudden microbial changes in intestine. Postwening intestinal microbiota dysbiosis is associated with some unstable situation including enteric infectious pathogens growth, leaky gut, and incomplete intestinal integrity [4].
Besides, in hot seasons, weanling pigs are under the negative influence of high temperatures, which can dramatically enhance the adverse effects of weaning stress. The low recovery rate of heatstressed weanling piglets is mainly due to the increased oxidative damage [5]. These critical conditions impair the immune system function and intestinal mucosal development, making the piglets susceptible to oxidative stress and microbial invasion.
Supplementation of pharmacological doses of ZnO (2500 ppm or over) in the piglet diet is a common practice among feed mills and farmers to control the adverse effects of postweaning diarrhea. There is evidence that high doses of dietary ZnO allow a higher tolerance to pathogen growth in the intestine [6]. Besides, dietary supplementation with high doses of ZnO increases the absorption rate that can enhance the antioxidant status and decrease the inflammatory responses in organs [7].
However, feeding high doses of ZnO results in substantial quantities of Zn excretion and increasing environmental concern. In order to reduce Zn excretion, the use of pharmacological doses of ZnO is banned and a lower concentration of ZnO is recommended in most of the developed countries.
It is well documented that the infectious morbidity attenuates by L-arginine (Arg) as an immunenutrient factor. The protective role of Arg is related to the protection of intestinal mucosa and the increase in the recovery rate in the intestinal barrier [8,9]. The nutritional requirement of Arg is highly dependent on environmental and physiological conditions. Therefore, stressors such as microbial change, heat stress, and sepsis dramatically increase the Arg requirements [10][11][12].
Arginine plays a crucial role in the production of polyamine and NO, which are strong anti-stress and vasodilator factors in mammals [2]. In addition, Arg facilitates the provision of cellular signaling, intestinal recovery, and immunity factors [10,13]. Therefore, it can be hypothesized that the use of high dietary Arg levels may decrease the ZnO requirement during the stressful condition. Little information is available on the role of Arg and ZnO on oxidative damage of heat-stressed weanling piglets, in particular, that of immunity status, intestinal integrity, and growth performance. In this study, high dietary Arg and different levels of ZnO are applied to evaluate their effects on growth performance, immune status, intestinal microbiota and morphology of weaned pigs.

Materials And Methods
The project underwent proper ethical standards and the experiments (KW-170519-1) were approved by the Institutional Animal Care and Use Committee of Kangwon National University, Chuncheon,

Republic of Korea.
Animals and Experimental Design. A total of 180 weaned pigs (28 day-old; Landrace × Yorkshire × Duroc; initial BW: 10.45 ± 0.03 kg) of mixed sex were randomly allotted to six treatments with 6 replicate pens in each treatment and 5 pigs per pen. The dietary treatments were: control diet (Con; with 1.1% Arg and without ZnO supplementation); Con + 2500 ppm Zn as ZnO (P-Zn); CON + 1.6% Arg (ARG); Con + 500 ppm of Zn as ZnO + 1.6% Arg (ZnArg1); Con + 1000 ppm of Zn as ZnO + 1.6% Arg (ZnArg2); P-Zn + 1.6% Arg (ZnArg3). All diets (Table 1) met or exceeded the nutrient requirements according to the NRC (2010). The crude protein, ether extract, lysine, methionine, arginine, calcium, and phosphorus content of the diets were analyzed by methods of AOAC [14]. The treatment diets were fed in a meal form in 2 phases (d 0 to 7, phase Ⅰ; and d 8 to 14, phase Ⅱ). This experiment was conducted at the facility of Kangwon National University farm and the piglets were housed in slotted and concrete floor pens with a pen size of 1.90 m × 3.0 m. All pens were equipped with a self-feeder and nipple drinker to allow ad libitum access to feed and water. Individual weanling piglet weight and feed intake from each pen were recorded at the beginning of the experiment and at the end of every phase to calculate average daily gain (ADG), average daily feed intake (ADFI) and gain to feed ratio (G:F). All the weaned pigs were subjected to a mild heat stress condition at 35° C.

Microbial analyses.
To study the effects of dietary treatments on small intestinal microbiota, representative piglets from each group (2 piglets per replicate; one male and one female) reflecting the average BW of the pen were selected and sacrificed by electrocution at d 14 and 28 of each phase. The digesta from the ileum and was collected in sterile plastic bottles for microbial analysis.
The samples collected for microbial analysis were immediately placed on ice until analyses were conducted. The ileal digesta sample (one gram) was mixed with 9 ml peptone broth (1%) and the Small intestinal morphology. The sacrificed pigs (two pigs per pen) were subjected to use for the morphological test. The intestinal morphology test was performed according to the procedure described by Hosseindoust et al., [15]. In short, for each intestinal sample, three cross-sections were prepared after staining with azure A and eosin using standard paraffin-embedding procedures. A total of 10 intact, well-oriented crypt-villus units were selected in triplicate for each intestinal cross-section.
The measurement of villus height was measured from the tip of the villi to the villus-crypt junction, while the crypt depth was defined as the depth of the invagination between adjacent villi and villus width was measured till the mid of the villus. All morphological measurements (villus height and crypt depth) were made in 10-μm increments using an image processing and analysis system (Optimus software version 6.5, Media Cybergenetics, North Reading, MA, USA).

RNA Extraction and Real-time PCR of organ samples.
Total RNA was isolated from the Jejunum (50 mg), livers (50 mg) and spleens (100 mg) samples using Trizol reagent (Invitrogen, Carlsbad, USA) according to manufacturer's instruction. Extracted RNA was quantified to 1 μg/μl and cDNA synthesis was conducted using the Improm-II Reverse transcription system (Promega, Fitchburg, USA) and PCR was performed using Mx3000P real-time PCR (Stratagen, USA). The results were expressed as a relative expression by using the delta-delta method. The primers of interleukin-4 (IL-4), interleukin-6 (IL-6), interferon-γ (IFNγ), heat shock protein-27 (HSP27), toll-like receptor-4 (TLR4), and tumor necrosis factor-α (TNF-α) were presented in Table 2. In this process, the house-keeping gene, βactin was introduced to adjust the quantity of input cDNA to maintain the role in internal control [16]. Statistical Analysis. The data were analyzed as a completely randomized design using the GLM procedure of SAS (SAS Inst. Inc., Cary NC). The pen was the experimental unit for growth performance and feed intake, whereas individual piglet was an experimental unit for the microbial test, intestinal morphology, blood parameters, and gene expression analyses. The Turkey multiple range tests were applied for Treatment means separation by at P < 0.05 statistical level.

Growth performance
The results of growth performance are shown in Table 3. There was no difference in the ADG of pigs in the first and second phases. The overall result showed that the inclusion of ZnArg3 significantly improved (P < 0.05) the ADG compared with the Con treatment. There was a reduction of ADFI in the Con diet compared with the ZnArg3 diet in phase 1 and overall. The gain to feed ratio was not affected by the treatments.

Microflora composition
The ileal microbial population is shown in Table 4. There was no difference in the population of Total anaerobic bacteria, Bifidobacterium spp., Lactobacillus spp., and Coliforms between the treatments.
At phase 1, the weaned pigs in the ZnArg3 and P-Zn groups exhibited the decreased population of Clostridium spp. in the ileum compared with those of the Con group (P < 0.01). In addition, a lower ileal Clostridium spp. population was detected in the ZnArg2 pigs compared with the Con pigs. At phase 2, the colonization of Clostridium spp. was higher in the Con, and ARG treatments compared with ZnArg3 treatment.

Intestinal morphology
As shown in Table 5, when the weaned pigs fed the ZnArg1 and ZnArg3 diets the villus height of Duodenum was increased compared with the Con and P-Zn treatments. There was no difference in villus height in the jejunum and ileum. In addition, the crypt depth and villus height to crypt depth ratio were not affected by supplementing Arg or ZnO in the diet.

Blood parameters
The results of the blood parameters are shown in Table 6. The number of WBC and RBC was not affected by the treatments. The number of Lymphocytes was not affected at d 7, however, there was a lower number of lymphocyte in the treatments ZnArg1, ZnArg2, and ZnArg3 compared with the Con pigs at d 14. There was no difference between the treatments for the number of monocytes, however, eosinophil number was significantly higher in the Con and ZnArg1 compared with the ZnArg2 and ZnArg3 treatments. Plasma cortisol was not affected by the treatments.

Gene expression in the organs
The weaned pigs in the Con group showed an increased mRNA expression of HSP27 in the liver compared with the P-Zn, ZnArg1, ZnArg2, and ZnArg3 groups (P < 0.05, Figure 1). An increased gene expression of TLR4 was observed in the ARG pigs compared with ZnArg3 pigs. There were no differences between the treatments in the gene expression of IL-4, IL-6, IFNγ, and TNF-α in the liver.
When fed the basal diet, weaned pigs exhibited enhanced mRNA expressions of IL-6 in the muscle compared with the ZnArg3 group (P < 0.05, Figure 2). Dietary supplementation with ZnArg2 decreased the mRNA expressions of IFNγ in the muscle compared with the Con group (P < 0.05).
Supplementation with P-Zn, ZnArg1, ZnArg2, and ZnArg3 exhibited decreased mRNA expressions of TNF-α compared with the Con group (P < 0.05). There were no differences between the treatments in the gene expression of IL-4, HSP27, and TLR4. The mRNA gene expressions of IL-4 were decreased in the jejunum of P-Zn, ARG, ZnArg1, ZnArg2, and ZnArg3 weaned pigs compared with the Con group ( Figure 3; P < 0.05). The ZnArg1, ZnArg2, and ZnArg3 treatments showed a lower mRNA expression of TNF-α compared with the Con group. The gene expression of TLR4 in the jejunum was upregulated in the Con and ARG treatments compared with the ZnArg1 and ZnArg3. The treatments did not significantly impact the mRNA expression of IL-6, IFNγ, and HSP27 in the jejunum.

Discussion
The supplementation of pharmacological doses of ZnO is routinely considered in weaned pigs diet to alleviate diarrhea incidence and reduce the growth depression during the weaning period [6,17]. In our study, the response to ADG and ADFI in pigs supplemented with ZnArg3 was increased relative to the Con pigs, however, there were no differences between the Arg and ZnO supplemented treatments. Compromising the growth performance in the Con pigs could be resulting from the insufficient nutrients intake, as shown by the significantly reduced ADFI in the Con pigs. The finding that supplementing 2500 mg/kg of Zn as ZnO showed no growth benefit compared with 500 or 1000 mg/kg Zn as ZnO was in contrast to previous researchers, who reported a significant difference between 2500 mg/kg ZnO and levels less than 1000 mg/kg [18]. It is believed that the lower doses of ZnO (below 1000 ppm) is not effective in the performance of weaned pigs [6,19]. Our results showed that there was a comparable ADG between the ZnArg1, ZnArg2, and ZnArg3. It seems that the  [7] reported that the antimicrobial activity of ZnO against coliforms makes it an ideal candidate to control the microbiota, however, they did not report any change in the number of Clostridium spp. In another study, the population of coliforms and Clostridium spp. was linearly decreased in the ileum and colon of weaned pigs when the dose of ZnO increased from 500 ppm to 2500 ppm [6]. It is possible to speculate that influences on the intestinal microbiota might not be modified by the doses lower than the pharmacological recommendation for ZnO. Also, the addition of an anti-stress nutritional item such as Arg did not aid the antimicrobial effects. This result disagrees with the report of Ren et al.
[23], who supplemented a high dose of Arg into the diet of mice to manipulate the microbial change in favor of beneficial bacteria including Lactobacillus spp. It is crucial to investigate whether Arg alone or a combination with ZnO in lower doses, will influence the weaned pig's microbiota and immune status.
In the present study, the combination of heat stress and weaning challenge in untreated pigs caused severe morphological disruption of villus that was shown by a decreased villus height in the duodenum of the Con pigs compared with ZnArg1 and ZnArg3 pigs. Intriguingly, the villus height in the jejunum and ileum remained unchanged. This result was surprising as the authors had

Gene
In the present study, the supplementation of ZnO remarkably decreased the mRNA expression levels of HSP27 that was involved in liver inflammation. The association between HSP expression and stress was previously reported in other studies [2,4]. The low gene expression of HSP27 in the liver and a tendency for lower blood cortisol in Zn-supplemented diets may indicate a lower stress level, however, the pigs fed high dietary Arg did not show any improvement in reducing the stress level, although Arg is reported to be an anti-stress or immunomodulatory factor by the activation of ornithine decarboxylase and generating polyamines [10]. In addition, the reports related to the influence of dietary Arg on the TLR gene expression in weanling pigs during heat stress is scanty. In       Table 6. Effect of heat stress on blood characteristics in weanling piglets fed diets supplemented with or without zinc and arg