Effects of dietary xylo- oligosaccharide on growth performance, serum biochemical parameters, antioxidant function, and immunological function of nursery piglets

This study investigated the effects of dietary xylo-oligosaccharide (XOS) on growth performance, serum biochemical parameters, antioxidant function, and immunological function of nursery piglets. In total, three groups including 72 nursery piglets were designed and fed one of three diets: a control basal diet, basal diet supplemented with 0.2% ZnO, or basal diet supplemented with 0.04% XOS, for 28 days. Compared with the control group, the XOS group significantly increased the final body weight and average daily weight gain. No significant differences were found about these parameters between the control and ZnO groups. Compared with the control group, the ZnO group showed no changes in the serum content of total protein (TP), albumin (ALB), albumin:globulin (ALB:GLB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), glucose, triglyceride, total cholesterol (TC), or in the serum activity of amylase and alkaline phosphatase. However, in the XOS group, serum glucose content increased and blood urea nitrogen and triglyceride content decreased significantly. Compared with the control group, dietary supplementation with XOS significantly increased the serum activity of total antioxygenic capacity, superoxide dismutase, and catalase and decreased the serum activity of malondialdehyde. At the same time, serum IgG content in XOS group was significantly higher than that in control group. From the current study, supplementation of 0.04% XOS in the diet could improve the antioxidant and immune function of piglets, promotes nitrogen deposition, and accelerates lipid and glucose metabolism, thereby improving piglet growth performance.


Introduction
Weaning is critical to pigs due to physiological and immunological immaturity that has been associated with increased susceptibility to infection and disease, especially diarrhea, and reduced growth performance (Campbell et al., 2013).Thus, the use of feed additives has become a common practice as modulator of immune system and intestinal microbiota, thereby promoting health and performance of pigs during the immediately post-weaning period (Lallès et al., 2007). Pharmacological levels of Zn oxide (ZnO), above 2000 mg ZnO/kg, have been widely used to control diarrhea (Owusu-Asiedu et al., 2003) as well as to improve feed efficiency and growth performance in weanling pigs (Walk et al., 2015;Payne et al. 2006). These beneficial effects of ZnO supplemented in the diet are effective until up to three weeks after weaning; however, some authors (Janczyk et al., 2013;Starke et al., 2014) observed the adverse effects due to toxicity. Because of its low digestibility, about 80% of ZnO is expelled through the The European Union has voted for the phase-out pharmacological ZnO by 2022 . The Chinese government has also explicitly proposed that all drug feed additives should be withdrawn from livestock and poultry breeding by 2020 (Ministry of Agriculture and Rural Areas of the People's Republic of China, 2018). Therefore, it has become a major research topic to investigate preferable alternative growth promoters.
Xylo-oligosaccharide (XOS) is a functional oligosaccharide that consists of two to seven xylose molecules linked by a beta-1,4 glycoside bond. It can pass through the stomach and small intestine, travels directly into the hindgut, then is utilized by beneficial bacteria in the gut (Jacobsen and Wyman, 2002). It is also fermented into short-chain fatty acids (SCFA) such as acetic, propionic, butyric, and valeric acids. These SCFA can reduce intestinal pH, inhibit the growth of harmful bacteria, regulate the intestinal flora balance, and protect intestinal health (Zhao et al., 2018). Xylo-oligosaccharide is widely used in healthy animal husbandry breeding and has become a new type of green feed additive due to its many advantages, such as safety, effectiveness, and low toxicity. In July 2003, XOS was first certified as a new feed product and feed additive by the Ministry of Agriculture of China (Announcement of the Ministry of Agriculture of the People's Republic of China,No. 288). Many studies (Wang and Lu, 2013;Yang et al., 2015;Jain et al., 2015) have showwn that it preferentially stimulates the growth or activity of beneficial bacteria (such as Bifidobacterium and other lactic acid bacteria) in the gut, enhances immune function, improves the growth of the intestinal mucosa, meanwhile increasing the microbiota diversity of the intestine (Tan et al., 2016) of weanling pigs.
However, little research has been performed regarding the application of XOS for nursery piglets. The purpose of the present study was to determine the effects of XOS on growth performance, serum biochemical parameters, antioxidant function, and immunological function of nursery piglets. This study aims to provide new insights for the wide application of XOS as a new type of green feed additive.

Material and Methods
The experiment was conducted in Yuanjiang (28°50'40.84" N, 112°20'55.60" E), Hunan province, China. All experimental procedures used in the present study were approved by the Animal Care Committee (case no. 2018009).
Seventy-two nursery pigs (Ningxiang pig, a local breed of Hunan, China; Castrate pig;average body weight [BW] = 18.94±0.24 kg) were randomly divided into three treatment groups. Each treatment consisted of four replicate pens and six pigs per replicate. Pigs were fed either a control basal diet, basal diet supplemented with 0.2% ZnO, or basal diet supplemented with 0.04% XOS. Each compound was uniformly mixed into the basal diet. These diets without antibiotics were formulated to provide the nutrients needed to meet or exceed the NRC requirements (Table 1) (NRC, 2012).
All diets were pelleted. Piglets were fed two times (8.00 and 18.00 h) every day. Access to feed and water were provided ad libitum throughout the experiment. Pretest period was seven days and experimental period was 28 days.
Piglets were weighed on day 1 and 28 of experimental period. Feed intake was recorded daily per pen to determine the average daily gain (ADG), average daily feed intake (ADFI), and feed intake to body gain ratio (F:G). A 10 mL of blood sample (three pigs per pen) was taken from the anterior vena cava of each animal on day 28 and centrifuged at 3 000 rpm for 10 min to collect serum (stored at −20 °C).
The experimental data were initially collated by Excel 2016 and then analyzed by one-way ANOVA with IBM SPSS Statistics 21.0 (IBM, Armonk, New York, US). The results were expressed as means. Significant means were separated by Duncan's Multiple Range test method. The significance level was P≤0.05, whereas 0.05 < P<0.1 was considered as a tendency of significant difference.

Results
The group fed XOS showed a significant increase in final BW and ADG (P<0.05) comparing to the control group (Table 2). No significant difference was observed in growth performance between the control and ZnO groups.
To evaluate the effects of XOS on blood composition, biochemical parameters (Table 3) were analyzed. Xylo-oligosaccharide significantly decreased (P<0.05) BUN and TG concentrations compared with the control group, but significantly increased (P<0.05) the GLU concentration compared with with control and ZnO groups. There were no significant effects on any other biochemical parameters.
Compared with the control group, serum T-AOC, SOD, and CAT activities were significantly higher (P≤0.05), but serum MDA activity was significantly lower (P<0.05) in the groups fed ZnO and XOS (Table 4). Dietary addition of XOS had a tendency to increase GSH-Px (P = 0.061) and IgG content in serum (P = 0.097).   Effects of dietary xylo-oligosaccharide on growth performance, serum biochemical parameters, antioxidant... Hou et al. 5

Discussion
Xylo-oligosaccharide plays a beneficial role in intestinal function by targeting tight junction proteins (Nawaz et al., 2018) and exhibits a probiotic effect by promoting the proliferation of beneficial microbes (Ho et al., 2018). Therfore, XOS is widely used as a probiotic to promote animal growth in animal production. Dietary supplementation of 0.01% XOS (Tan et al., 2016) could significantly increase ADG and ADFI in weanling pigs. Similarly, dietary supplementation with 20 mg/kg XOS in weanling piglets gained in a greater ADG, ADFI, and FCR (Zhao et al., 2018). Some researchers reported that inclusion of 200 mg/kg XOS in diet significantly improved the growth performance of nursery pigs (Fang et al., 2015;Fan et al., 2016;Guo et al., 2017;Liu et al., 2018). The current study obtained similar results that dietary supplementation with 0.04% XOS for nursery pigs could significantly improve the final BW and ADG (P<0.05). However, the present results were contradictory to the literature reported, in which supplementary 0.01% XOS affected growth performance weakly (Yin et al., 2019). This may be due to the growth stage of the pigs, as well as different sources and dosage of XOS.
High serum TP content is beneficial for enhancing the metabolic function and immunity of animals. A decreased ALB:GLB indicates (Sun et al., 2009) that the synthesis of immunoglobulin is accelerated, accompanied by enhanced disease resistance. Researchers (Zhao et al., 2018;Yin et al., 2019) reported that XOS failed to affect serum TP, ALB, or ALB:GLB in pigs. The results of this study showed that diet supplementation with XOS for nursery piglets did not significantly affect serum TP, ALB, and ALB:GLB. However, serum TP content was higher and ALB:GLB was lower in XOS-fed pigs.
The present results may have been due to the breed of the piglets used, which could easily adapt to feeding management conditions and have strong anti-stress mechanisms.
Alanine aminotransferase and AST are two important amino acid transferases found in the mitochondria and cytoplasm of hepatocytes. When animals are damaged by liver injury or acute stress, the serum activities of these two enzymes will increase (Chen et al., 1995). Therefore, the activity of ALT and AST in the serum can reflect damage to hepatocytes (Lv et al., 2015;Liu et al., 2015). Tan et al. (2016) reported that the activities of AST in plasma of 500 g/t XOS group were significantly higher than those of positive control group on day 7 and 56 of experiment, and the activity of ALT in plasma of 100 and 500 g/t XOS groups was significantly higher than that of blank control group on day 21 of experiment.
As the number of XOS addition increases, the activities of ALT and AST in serum had a tendency to improve, but there were no significant differences among all groups (Zhao et al., 2018). Yin et al. (2019) found that supplementation with XOS in diet did not affect the serum activities of ALT and AST. The results of this experiment were consistent with Yin et al. (2019), in which no significant differences were observed in the serum activities of ALT and AST in the nursery piglets among groups, indicating that the piglets had strong stress resistance and XOS added in the diet had no harmful effects on their livers. This is contrary to other findings (Tan et al., 2016;Zhao et al., 2018) because it may be due to pig breed or XOS types and doses, etc.
A previous study reported that the appropriate dietary supplementation with XOS could improve serum GLU content (Singh and Cresswell, 2010). Some researchers found that the addition of XOS to the pig diet could significantly decrease serum BUN and TG contents (Pan, 2011;Tan et al., 2016;Zhao et al., 2018). In the present study, it was found that the addition of XOS in diet could significantly increase GLU contents and reduce the contents of serum BUN and TG. Under normal conditions, the serum GLU content of high-yield animals has been found to be higher than that of low-yield animals (Hou et al., 2015), which reflects the glucose metabolism status of the body. The serum content of BUN is an important indicator of protein synthesis efficiency and amino acid balance (Coma et al., 1995), which is negatively correlated with muscle growth and ADG in piglets. Serum TG levels are known to increase when the body is under stress, which could result in decreasing fat utilization (Chen et al., 2016). Our results showed that XOS added to the diet could enhance the digestion and absorption of nutrients, increase the efficiency of protein biosynthesis and fat utilization, and simultaneously reduce adverse stress reactions in piglets and improve their health status.
Activity of AMY reflects the digestive and absorptive capacity of starch and affects the speed of chemical reactions, growth, developmental health, and the adaptability of the body. Furthermore, AKP promotes the deposition of calcium and phosphorus in bone and also regulates the metabolic functions of the body. Pan (2011) reported that adding 0.02% XOS in diet significantly increased the activities of AKP and AMY. The activity of AMY in plasma of group fed 500 g/t XOS was significantly higher than that of positive control group on day 56 of experiment (Tan et al., 2016). In another study, it was found that the activities of AMY and AKP in serum of dietary XOS (200 mg/kg) group for 28 days in weaned pigs were significantly higher than those of control group (Zhao et al., 2018). The results of this study showed that the 0.04% XOS addition to diet had a tendency to increase the activity of AMY and AKP in serum. However, no significant differences were observed among groups in the current study, which were contrary to previous results (Pan, 2011;Zhao et al., 2018), possibly because of the duration of this experiment and growth stages of the piglets, as well as the composition and source of the diet.
Serum T-AOC is an indicator of the overall level of enzymatic and non-enzymatic antioxidants in vivo. Enzymatic oxygen free radical scavengers SOD, CAT, and GSH-Px are the major components of the enzymatic antioxidant system. Malondialdehyde is a metabolic product of lipid peroxidation and indirectly reflects the degree of damage by oxygen free radicals to cells. When the concentration of free radicals increases in the body, tissues will enhance the endogenous activity of the antioxidant system to prevent free radical damage. Fan et al. (2016) found that the diets supplemented with 200 mg/kg XOS markedly enhanced the serum T-AOC, liver SOD, and CAT activity and decreased MDA contents of serum and liver. From the present results, dietary supplementation with 0.04% XOS significantly improved serum T-AOC, SOD, and CAT activities and significantly decreased serum MDA activity. Therefore, under this experimental condition, the results indicated that 0.04% XOS added to diet could enhance the antioxidant capability in vivo.
Serum Ig is a non-specific antibody found in animal serum. It mainly plays a protective role in the immune systems, as they are stimulated by the invasion of foreign pathogens and viruses to protect the body against infection. Supplementation of XOS in pig diets could significantly increase serum IgG content (Fang et al., 2015;Pan, 2011;Wang et al., 2006). The current study showed similar results, as dietary supplementation with XOS could improve the serum IgG content to a certain extent. This result showed that the suitable addition of 0.04% XOS in diet might have anti-infective effect which needs to be confirmed with further experiments.

Conclusions
Under the current experiment conditions, dietary 0.04% xylo-oligosaccharide supplementation could improve the antioxidant and immune function of piglets, promote nitrogen deposition, and accelerate lipid and glucose metabolism, thereby improving their growth performance.