Scholarly article on topic 'Effects of spermine supplementation on the morphology, digestive enzyme activities, and antioxidant capacity of intestine in weaning rats'

Effects of spermine supplementation on the morphology, digestive enzyme activities, and antioxidant capacity of intestine in weaning rats Academic research paper on "Animal and dairy science"

CC BY-NC-ND
0
0
Share paper
Academic journal
Animal Nutrition
OECD Field of science
Keywords
{"Antioxidant capacity" / "Enzymes activities" / Morphology / Spermine / "Weaning rats"}

Abstract of research paper on Animal and dairy science, author of scientific article — Tingting Fang, Gang Jia, Hua Zhao, Xiaoling Chen, Jiayong Tang, et al.

Abstract The main objective of this study was to investigate the effects of different doses of spermine and its extended supplementation on the morphology, digestive enzyme activities, and intestinal antioxidant capacity in weaning rats. Nineteen-day-old male rats received intragastric spermine at doses of 0.2 and 0.4 μmol/g BW for 3 or 7 d, whereas control rats received similar doses of saline. The results are as follows: 1) In the jejunum, the seven-day supplementation with both doses of spermine significantly increased crypt depth (P < 0.05) compared with the control group; the supplementation extension of the high spermine dose increased villus height and crypt depth (P < 0.05); in the ileum, the low spermine dose significantly increased villus height and crypt depth compared with the control group for 7 days (P < 0.05). 2) The 3-day supplementation with high spermine dose increased alkaline phosphatase activity in the jejunum (P < 0.05). 3) In the jejunum, the anti-hydroxyl radical (AHR), total superoxide dismutase (T-SOD), catalase (CAT), and total antioxidant capacity (T-AOC) activities were increased (P < 0.05); however, the malondialdehyde (MDA) content was reduced (P < 0.05) in groups supplemented with the high spermine dose relative to those in the control groups after 3 and 7 d; moreover, the anti-superoxide anion (ASA) and glutathione (GSH) contents increased with the high spermine dose that lasted for 3 days (P < 0.05). Furthermore, the T-SOD and CAT activities (after 3 and 7 d), ASA (after 3 d), and AHR (after 7 d) increased with the high spermine dose compared with those of the low spermine dose (P < 0.05). Extending the supplementation duration (7 d) of the high spermine dose decreased the MDA content and ASA and T-AOC activities (P < 0.05). These results suggested that spermine supplementation can modulate gut development and enhance the antioxidant status of the jejunum in weaning rats, and a dosage of 0.4 μmol spermine/g BW had better effects than the dosage of 0.2 μmol spermine/g BW on accelerating gut development and increasing antioxidant capacity.

Academic research paper on topic "Effects of spermine supplementation on the morphology, digestive enzyme activities, and antioxidant capacity of intestine in weaning rats"

Animal Nutrition

" KeAi fe

Accepted Manuscript

Effects of spermine supplementation on the morphology, digestive enzyme activities, and antioxidant capacity of intestine in weaning rats

Tingting Fang, Gang Jia, Hua Zhao, Xiaoling Chen, Jiayong Tang, Jing Wang, Guangmang Liu

PII: S2405-6545(16)30138-X

DOI: 10.1016/j.aninu.2016.09.002

Reference: ANINU 116

To appear in: Animal Nutrition Journal

Received Date: 4 August 2016 Revised Date: 31 August 2016 Accepted Date: 18 September 2016

Please cite this article as: Fang T, Jia G, Zhao H, Chen X, Tang J, Wang J, Liu G, Effects of spermine supplementation on the morphology, digestive enzyme activities, and antioxidant capacity of intestine in weaning rats, Animal Nutrition Journal (2016), doi: 10.1016/j.aninu.2016.09.002.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effects of spermine supplementation on the morphology, digestive enzyme

activities, and antioxidant capacity of intestine in weaning rats

3 Tingting Fanga b, Gang Jiaa b, Hua Zhaoa b, Xiaoling Chena, b, Jiayong Tanga, b,

5 a Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu 611130,

6 China

7 b Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of

8 Education, Chengdu 611130, China

9 c Maize Research Institute, Sichuan Agricultural University, Chengdu 611130, China

Jing Wangc, Guangmang Liu

a, b, *

14 Corresponding author.

15 E-mail address: liugm@sicau.edu.cn (G. Liu)

ABSTRACT

The main objective of this study was to investigate the effects of different doses of

spermine and its extended supplementation on the morphology, digestive enzyme

activities, and intestinal antioxidant capacity in weaning rats. Nineteen-day-old male

rats received intragastric spermine at doses of 0.2 and 0.4 [j,mol/g BW for 3 or 7 d,

whereas control rats received similar doses of saline. The results are as follows: (1) In

the jejunum, the seven-day supplementation with both doses of spermine significantly

increased crypt depth (P < 0.05) compared with the control group; the

supplementation extension of the high spermine dose increased villus height and crypt

depth (P < 0.05); in the ileum, the low spermine dose significantly increased villus

height and crypt depth compared with the control group for 7 days (P < 0.05). (2) The

3-day supplementation with high spermine dose increased alkaline phosphatase

activity in the jejunum. (3) In the jejunum, the anti-hydroxyl radical (AHR), total

superoxide dismutase (T-SOD), catalase (CAT), and total antioxidant capacity

(T-AOC) activities were increased; however, the malondialdehyde (MDA) content

was reduced in groups supplemented with the high spermine dose relative to those in

the control groups after 3 and 7 d; moreover, the anti-superoxide anion (ASA) and

glutathione (GSH) contents increased with the high spermine dose that lasted for 3

days (P < 0.05). Furthermore, the T-SOD and CAT activities (after 3 and 7 d), ASA

(after 3 d), and AHR (after 7 d) increased with the high spermine dose compared with

those of the low spermine dose (P < 0.05). Extending the supplementation duration (7

d) of the high spermine dose decreased the MDA content and ASA and T-AOC activities (P < 0.05). These results suggested that spermine supplementation can modulate gut development and enhance the antioxidant status of the jejunum in weaning rats, and a dosage of 0.4 [j,mol spermine/g BW had better effects than the dosage of 0.2 [j,mol spermine/g BW on accelerating gut development and increasing antioxidant capacity.

Keywords: Antioxidant capacity, Enzymes activities, Morphology, Spermine, Weaning rats

1. Introduction

The intestine plays a crucial role in digesting and absorbing nutrients, balancing microbiota, protecting immunological functions in young animals and serves as a barrier against harmful pathogens and antigens (Lalles et al., 2007; Barszcz and Skomial, 2011). Weaning is one of the most complex and stressful periods that may lead to intestinal dysfunction, resulting in increased susceptibility to diseases, dyspepsia, and diarrhea, all of which can degrade animal health and growth after weaning (Lalles et al., 2007). Thus, maintaining the normal functions of the small intestine and promoting intestinal maturity is essential for animal growth and development after weaning in livestock production. Various experiments have been conducted to prove that nutritional regulation is feasible and can facilitate intestinal development during weaning periods (Kim et al., 2004; Wang et al., 2015).

Spermine, is found in nearly all tissues and cells as a low-molecular-weight molecule, and the main source of spermine in dietary are corn, rice, cheese, fruit, meat, and some vegetables (Larque et al., 2007). Previous studies have shown that spermine is an essential nutrient that takes part in multiple cellular and physiological processes. Spermine can alleviate intestinal dysfunction and promote intestinal maturation in animals (Cao et al., 2015; Fang et al., 2016). Oral administration of spermine to suckling animals can induce structural and functional maturation in the small intestine, which is represented by morphological, enzymatic, and physiological alterations (Peulen et al., 2000). Spermine-induced morphological maturation is reflected by increased villus height, width, and crypt extension, all of which can help young animals adapt efficiently from the highly digestible and palatable liquid milk to a less digestible and palatable solid dry diet (Cheng et al., 2006;Campbell et al., 2013). Spermine can also affect enzymatic activities that accompany the morphological changes in the small intestine. Oral administration of spermine results in increased maltase- and sucrase-specific activities, but decreased lactase-specific activities in the jejunum and ileum of neonatal rats (Peulen et al., 2004; Liu et al., 2015). Previous studies suggested that oral administration of spermine for 3 days after weaning enhances the development of the small intestine in weaning pigs (Kang et al., 2012). Nevertheless, few studies have been conducted on the effects of prolonged spermine administration on the intestinal development of weaning animals.

Moreover, spermine protects the organs from oxidative damage. Spermine can change

the antioxidant status of the jejunum by scavenging free radicals in lactating rats, and can also mitigate serum oxidative stress in weaning rats (Liu et al., 2014a, 2014b; Cao et al., 2015). Spermine can also maintain the redox balance in suckling piglets by enhancing the antioxidant and non-antioxidant enzyme activities in the serum (Fang et al., 2016). These findings indicate that spermine is capable of enhancing antioxidant effects in vivo. However, no information is available on the effects of different spermine doses and its extended supplementation on the antioxidant properties in weaned animals, therefore there is a need for further investigation.

This study is part of a larger study that investigates the metabolomic effects of spermine administration against weaning stresses in rats (Liu et al., 2014b). This study aimed to evaluate the effects of different doses of spermine supplementation and its extended administration on intestinal development and antioxidant status of the jejunum in weaning rats. We investigated the changes in histomorphological structures and digestive enzyme activities in the jejunum and ileum, as well as the antioxidant parameter alterations of the jejunum in weaned rats subjected to spermine administration. The results of this work could provide scientific evidence for future studies on the relationship between spermine supplementation and extended spermine administration on intestinal health.

2. Materials and methods

2.1. Materials

Spermine (S3256-1G) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Sprague-Dawley rats with a body weight (BW) from 38 to 45 g were provided by Dossy Experimental Animals Co., Ltd (Chengdu, China). All enzyme assay and antioxidative reagent kits used in the present study were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

2.2. Animal experiment and sample collection

Animal experimental procedures of this work were approved by the Care and Use of

Laboratory Animals of Sichuan Agricultural University of China. All protocols were

conducted according to the Guide for the Care and Use of Laboratory Animals of the

National Research Council (1997). Thirty six 19-day-old weaning male

Sprague-Dawley rats were placed in individual metabolic cages and acclimatized for

1 d. All rats were randomly divided into 6 experimental groups (i.e., C-3, C-7, SO.2-3,

SO.2-7, SO.4-3, and SO.4-7) with 6 replicates per group and ad libitum access to diet

and water. The rats were intragastrically administered with either spermine (0.2 or 0.4

[j,mol/g BW) or physiological saline once a day for 3 or 7 d. Room temperature and

humidity were set to 25 °C and 60%, respectively, and a cycle of 12-h light and 12-h

dark was maintained throughout the experiment. Rats were anesthetized by ether 48 h

after the last spermine ingestion. The jejunum and ileum were separated and

immediately flushed with ice-cold saline. The isolated jejunal and ileal segments of

approximately 3 cm in length were stored in 4% paraformaldehyde solution for

morphological analyses. The remaining jejunum and ileum samples (approximately 5

cm) were snap-frozen in liquid nitrogen and then stored at -80 °C before analysis. The dosages and times of spermine supplementation were selected following a previous experiment (Deloyer et al., 2005).

2.3. Histomorphological study of the jejunum and ileum

The preserved tissues were sectioned and stained with hematoxylin and eosin, and applied with standard paraffin-embedding procedures. At least 10 intact, well-oriented crypt-villus units were selected in triplicate as sources of each rat intestinal cross section. Villus height, villus width, and crypt depth were measured using image processing and analysis systems (Image Pro Plus, Media Cybernetics, Bethesda, MD, USA). The villus surface areas were computed using the following formula: Villus surface area (mm ) = 2n (villus height) x (villus width/2).

2.4. Enzyme assays of the jejunum and ileum

The jejunal and ileal samples were thawed, weighed, and homogenized (1:10, wt/vol) in 9 volumes of ice-cold physiologic saline. The homogenates were centrifuged at 3,000 x g for 10 min at 4 °C, the supernatants collected and enzyme activities analyzed. Total protein contents were assayed using the method described by Bradford et al. (1976); maltase, sucrase, and lactase activities were determined as described by Dahlqvist (1964); AKP activity was determined according to the method of Rosalki et al. (1984).

2.5. Determination of antioxidant index in the jejunum

To evaluate the prooxidant-antioxidant balance in the jejunum, we determined the malondialdehyde (MDA) and glutathione (GSH) contents, as well as the anti-superoxide anion (ASA), anti-hydroxyl radical (AHR), total superoxide dismutase (T-SOD), catalase (CAT), and total antioxidant capacity (T-AOC) activities. The methods of preparing supernatants of jejunal homogenates were described as above (enzyme assay procedure). The MDA content was detected as described by Livingstone et al. (1990); ASA and AHR activities were determined followed the protocol of Jiang et al. (2009). Total superoxide dismutase activity was determined using the method of Zhang et al. (2010), and CAT activity was assayed according to Aebi (1984). GSH content was measured according to Vardi et al. (2008), and T-AOC activity was estimated according to Miller et al. (1993).

2.6. Statistical analysis

All data were subjected to two-way analysis of ANOVAusing the GLM procedure of SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) and presented as means ± standard errors. The main factorial of the model included spermine level (0, 0.2, 0.4 [j,mol/g BW) and extension time (3 or 7 d). Statistical differences between means were determined by ANOVA and Duncan's multiple range was used to compare data among treatments. Statistically significant differences were considered as P < 0.05.

3. Results

3.1. Morphological observations of the jejunum and ileum

Tables 1 and 2 present the morphological indices of the jejunum and ileum. In the jejunum, the 7-day supplementation with spermine of both doses significantly increased crypt depth, i.e., C-7 vs S0.2-7 and C-7 vs S0.4-7, (P < 0.05), whereas the 7-day supplementation with spermine of high dose significantly increased villus height, i.e., C-7 vs S0.4-7, (P < 0.05). The supplementation extension of high spermine dose increased villus height and crypt depth, i.e., S0.4-3 vs S0.4-7, (P < 0.05). Moreover, no difference was observed between C-3 and C-7 groups. In the ileum, the low spermine dose significantly increased both villus height and crypt depth compared with those in the control group after 7 days (P < 0.05).

3.2. Enzyme activities in the jejunum and ileum

Enzyme activities in the jejunum and ileum are presented in Tables 3 and 4. In the jejunum, the 3-day supplementation of high spermine dose increased alkaline phosphatase activity (C-3 vs S0.4-3). However, the obtained value did not differ between C-7 and S0.4-7 groups. Spermine also had no effect on digestive enzyme activities in the jejunum and ileum regardless of dose and supplementation duration.

3.3. Antioxidant indicators in the jejunum

Table 5 presents the antioxidant indicators in the jejunum. Jejunum ASA, AHR, T-SOD, CAT, and T-AOC activities and GSH content were increased; however, MDA content was reduced in groups supplemented with high spermine dose relative to those in the control group after 3 days, i.e., C-3 vs S0.4-3, (P < 0.05). The low

spermine dose increased AHR, CAT, and T-AOC activities and GSH content compared with control group, i.e., C-3 vs S0.2-3, (P < 0.05). Moreover, AHR, T-SOD, and CAT activities were higher in S0.4-7 group compared with C-7 and S0.2-7 groups (P < 0.05). Extending the supplementation duration (7 d) for both low and high spermine dose groups also decreased MDA content (P < 0.05). The extended high spermine dose decreased ASA and T-AOC activities relative to the values in S0.4-3 groups.

4. Discussion

4.1. Effects of spermine dose and extended spermine supplementation on jejunal and ileal morphological structures

During weaning, the intestine structure and morphology of animals will experience profound modifications (Barszcz and Skomial, 2011). These modifications are achieved by villus proliferation and crypt hyperplasia, which results from both the faster rate of cell division, leading to increased villus length and width, and increased rate of cell renewal, resulting in deeper crypts (Cummins and Thompson, 2002; Madara, 2011; Noah et al., 2011). Increases in villus height and crypt depth significantly contribute to enlargement of the absorptive area of intestinal mucosa. Our results showed that the extended supplementation of the high spermine dose increased villus height and crypt depth. These data suggested that extending spermine supplementation can promote jejunal development by regulating morphological

201 structures, which were consistent with the results of previous studies, wherein

202 spermine accelerated jejunal maturation (Fang et al., 2016). However, no differences

203 were observed in villus width, villus height-to-crypt depth ratio, and villus surface

204 area of jejunum in spermine-supplemented rats, which were consistent with some

205 results, wherein spermine did not significantly affect the jejunal indices in suckling

206 rats (Cao et al., 2015). The low spermine dose significantly increased villus

207 height-to-crypt depth ratio of ileum compared with that in the control groups. This

208 situation implied that the effects of spermine administration on ileum development

209 were influenced by spermine dosage. Evidence confirmed that excess spermine has

210 adverse effects in promoting cells growth; however, low or optimum doses of

211 spermine have beneficial effects on intestinal development (Cheng et al., 2006;

212 Larque et al., 2007). Collectively, the effects of different spermine doses and its

213 extended supplementation can modulate intestinal morphological changes, especially

214 those administered with 0.4 [j,mol/g BW.

215 Morphological changes in the intestine after weaning are accompanied by changes in

216 the activities of brush border enzymes, such as lactase, sucrase, and maltase, which

217 serve as important digestive functions. Hence, we probed the effects of spermine and

218 its extended administration on intestinal enzymatic activity.

219 4.2. Effects of spermine dose and its extended supplementation on the enzymatic

220 activities in rat jejunum and ileum

After weaning, the intestinal environment changes drastically because of the replacement of highly digestible sow milk with solid food. The intestine has to adapt to the new type of food, leading to changes in enzymatic secretion and activity. The decrease in lactase and increase in maltase and sucrase activities are believed to be signs of small intestinal maturity in young animals. However, our study found that spermine had no effect on lactase, maltase, and sucrase activities in the jejunum and ileum, which was consistent with the results of Peulen et al. (2004), who reported that spermine did not affect lactase or sucrase activities in the jejunum, maltase or sucrase activities of the ileum in 21-day-old weaning rats. These changes might be associated with age. The intestine might become unresponsive to spermine with age; similarly, spermine could not be recommended to facilitate changes in intestinal enzymatic patterns in typical weaning rats. Furthermore, the other fact is that intestinal enterocytes could become insensitive to spermine supplementation when the intestine becomes capable of obtaining higher contents of exogenous spermine from a solid diet after weaning (Larque et al., 2007). Nevertheless, the higher spermine dose increased the specific activity of alkaline phosphatase in the jejunum in the 3-day spermine treatment. These results suggested that supplementing 0.4 [j,mol spermine/g BW for 3 days could maintain the adult enzymatic pattern of alkaline phosphatase in the jejunum. Our results demonstrated that spermine intake and extended supplementation only have limited effects on enhancing the digestive and absorptive enzymatic activities of the intestine in weaning animals.

4.3. Effects of spermine dose and time extension of spermine administration on the antioxidant status of jejunum

During weaning, animals suffer grievously from oxidative stresses induced by numerous environmental, social, physical, and psychological stressors, such as different temperature and humidity, different food source, and abrupt displacement (Campbell et al., 2013; Yin et al., 2013). Oxidative stress can lead to overproduction of reactive oxygen species (ROS), which can react at a high rate with most of the molecules in the cells and damage proteins, amino acids, and nucleic acids (Srivastava et al., 2006). Spermine has a potential effect against oxidative stress, and can better promote jejunum development in our study. However, no research on the effects of spermine and its extended administration on the antioxidant defense system of organs in weaning animals is available. Therefore, our study further explored the protective effects of spermine dose and its extended supplementation on the antioxidant status of jejunum in weaning rats. The jejunal antioxidant indices include MDA content and ASA, AHR, CAT, T-SOD, GSH, and T-AOC activities.

Excessive amounts of ROS can lead to lipid peroxidation in organisms. Malondialdehyde (MDA) is the most familiar breakdown product of lipid peroxidation, and its level is frequently used as a direct marker of lipid oxidative damage caused by ROS (Feng et al., 2014). The results of MDA in this study indicated that spermine significantly reduced MDA content in the jejunum, thereby suggesting that spermine could depress lipid peroxidation in weaning rats. These

findings were similar to previous findings in suckling animals (Cao et al., 2015; Fang et al., 2016). Both doses of spermine could also decrease MDA content regardless of treatment duration. Moreover, lower MDA contents were observed with prolonged spermine supplementation for both doses, indicating that extending spermine administration can improve the capacity of anti-lipid peroxidation in the rat jejunum. Based on the results of spermine preventing lipid peroxidation in the jejunum, we further determined the scavenging ability of spermine on superoxide anion (O2-) and hydroxyl radical (OH-). The superoxide anion and hydroxyl radical are 2 agents strongly involved in lipid peroxidation and oxidative damage in cells (Kohen and Nyska, 2002). In the present study, spermine significantly enhanced ASA and AHR activities compared with the control group, indicating that spermine administration can improve the free radical scavenging ability in the jejunum. Our results also showed that ASA and AHR activities in j ejunum increased with higher spermine dose compared with those of lower spermine dose. High spermine dose appears to enhance the function of radical scavenging in the jejunum. Such observations are in accordance with those in other studies that confirmed spermine to be mostly a free radical scavenger only at very high doses in vitro (Kafy et al., 1986).

Scavenging capacity for free radicals is related to enzymatic and non-enzymatic antioxidant defense systems, and these defense systems were measured to further identify the manner of spermine-induced inhibition of oxidative damage in the jejunum. Total superoxide dismutase and CAT are representative enzymatic

antioxidants in the body. Superoxide dismutase is one of the most important endogenous antioxidants and the first enzyme to respond to oxygen radicals, as well as offer protection against oxidative stresses (Winston and Di Giulio, 1991). Superoxide dismutase can also transform hydroperoxide and superoxide anions into H2O2 and O2 through enzymatic degradation (Winston and Di Giulio, 1991). Catalase has been labeled as an essential H2O2 defense that eliminates the toxicity of hydroxyl radicals and decomposes H2O2 into O2 and H2O (David et al., 2008). In this study, we found that jejunal T-SOD and CAT activities were significantly increased by spermine administration. These results suggested that spermine supplementation can enhance antioxidative capacities. Significant enhancements of T-SOD and CAT were also observed following the high spermine dose, which suggested that spermine supplementation has dose-dependent effect on enhancing T-SOD and CAT activities in weaning rats. Our results revealed that spermine can enhance antioxidant status through enzymatic systems in weaning rats.

Glutathione is a major non-enzymatic antioxidant, and is commonly used to evaluate organ antioxidative capability. Glutathione is the most abundant intracellular thiol-based antioxidant scavenger and acts as the first line of defense against oxidative stresses (Yang et al., 2013). Total antioxidant capacity (T-AOC) is often adopted as an important index to reflect the total antioxidant capacity of the body and comprehensive indicator of the redox status in the host system (Ren et al., 2012). In the current study, spermine supplementation improved T-AOC activity and GSH

305 contents in rat jejunum. These findings suggested that spermine can protect jejunum

306 from oxidative stress. However, the underlying mechanism of the spermine mediates

307 non-enzymatic antioxidant systems requires further investigation. Spermine

308 supplementation can improve the antioxidant status through non-enzymatic

309 antioxidant systems in weaning rats.

310 The results proved that spermine can inhibit lipid peroxidation, improve free radical

311 scavenging abilities, and enhance the activities of enzymes and non-antioxidant

312 enzymes of jejunum in weaning rats. Particularly, the 0.4 [jmol spermine/g BW

313 exerted better effect than 0.2 [j,mol spermine/g BW on increasing antioxidant capacity.

314 5. Conclusions

315 The results of this study suggested that extending spermine supplementation can

316 promote the growth of the jejunum because spermine affects its morphology.

317 Spermine can affect the morphology of the ileum as well as the alkaline phosphatase

318 activity of the jejunum in weaning rats. Spermine administration and its extended

319 supplementation can also promote antioxidant defenses in weaning rats in dose- and

320 time-dependent manners. Weaning rats supplemented with 0.4 [j,mol spermine/g BW

321 exhibited better intestinal development and increased antioxidant capacity than 0.2

322 [j,mol spermine/g BW. These findings provide a new framework for elucidating the

323 effects of spermine ingestion and extended spermine supplementation to advance

324 intestinal development and enhance antioxidant capacity in weaning rats.

Acknowledgments

This research was financially supported by the National Natural Science Foundation

of China (No.31301986), the Academy of Kechuang Feed Industry in Sichuan and

Specific Research Supporting Program for Discipline Construction in Sichuan

Agricultural University (to G. Liu). The authors would like to express their sincere

thanks to the ongoing help of their teammates.

References

Aebi H. Catalase in vitro. Methods enzymolo 1984; 105: 121-26.

Barszcz M, Skomial J. The development of the small intestine of piglets-chosen aspects. Anim Feed Sci Technol 2011; 20(1): 3-15.

Bradford MM. A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72(1): 248-54.

Campbell JM, Crenshaw JD, Polo J. The biological stress of early weaned piglets. J Anim Sci Biotechnol 2013; 4(19): 1-4.

Cao W, Liu GM, Fang TT, Wu XJ, Jia G, Zhao H, et al. Effects of spermine on the morphology, digestive enzyme activities, and antioxidant status of jejunum in suckling rats. RSC Adv 2015; 5(93): 76607-14.

Cheng ZB, Li DF, Xing JJ, Guo XY, Li ZJ. Oral administration of spermine advances

intestinal maturation in sucking piglets. J Anim Sci 2006; 82(05): 621-26.

Cummins AG, Thompson FM. Effect of breast milk and weaning on epithelial growth of the small intestine in humans. Gut 2002; 51(5): 748-54.

Dahlqvist A. Method for assay of intestinal disaccharidases. Anal Biochem 1964; 7(1): 18-25.

David M, Munaswamy V, Halappa R, Marigoudar SR. Impact of sodium cyanide on catalase activity in the freshwater exotic carp, Cyprinus carpio (Linnaeus). Pestic Bochem Phys 2008; 92(1): 15-18.

Deloyer P, Peulen O, Dandrifosse G. Intestinal effects of long-lasting spermine ingestion by suckling rats. Exp Physiol 2005; 90(6): 901-8.

Fang T, Liu G, Cao W, Wu X, Jia G, Zhao H, et al. Spermine: New insights into the intestinal development and serum antioxidant status of suckling piglets. RSC Adv 2016; 6(37): 31323-35.

Feng L, Zhao S, Chen G, Jiang W, Liu Y, Jiang J, et al. Antioxidant status of serum, muscle, intestine and hepatopancreas for fish fed graded levels of biotin. Fish Physiol Biochem 2014; 40(2): 499-510.

Jiang J, Zheng T, Zhou XQ, Liu Y, Feng L. Influence of glutamine and vitamin E on growth and antioxidant capacity of fish enterocytes. Aquacult Nutr 2009; 15(4): 409-14.

Kafy A, Haigh C, Lewis D. In vitro interactions between endogenous polyamines and superoxide anion. Agents actions 1986; 18(5-6): 555-59.

Kang P, Wang M, Hou Y, Yin Y, Ding B, Zhu H, et al. Effects of oral administration of spermine on the development of small intestine and growth performance of weaned pigs. J Anim Vet Adv 2012; 11(15): 2782-2787.

Kim SW, Mcpherson RL, Wu G. Dietary arginine supplementation enhances the growth of milk-fed young pigs. J Nutr 2004; 134(3): 625-30.

Kohen R, Nyska A. Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods of their quantification. Toxicol Pathol 2002; 30 (6): 620-50.

Lalles J-P, Bosi P, Smidt H, Stokes CR. Weaning—a challenge to gut physiologists. Livest Sci 2007; 108(1): 82-93.

Larque E, Sabater-Molina M, Zamora S. Biological significance of dietary polyamines. Nutr 2007; 23(1): 87-95.

Liu GM, Fang TT, Yan T, Jia G, Zhao H, Chen XL, et al. Systemic responses of weaned rats to spermine against oxidative stress revealed by a metabolomic strategy. RSC Adv 2014a; 4(100): 56766-78.

Liu GM, Fang TT, Yan T, Jia G, Zhao H, Huang ZQ, et al. Metabolomic strategy for the detection of metabolic effects of spermine supplementation in weaned rats. J

Agri Food Chem 2014b; 62: 9035-42.

Liu GM, Yan T, Fang TT, Jia G, Chen XL, Zhao H, et al. Nutrimetabolomic analysis provides new insights into spermine-induced ileum-system alterations for suckling rats. RSC Adv 2015; 5(60): 48769-78.

Livingstone D, Martinez PG, Michel X, Narbonne J, O'hara S, Ribera D, et al. Oxyradical production as a pollution-mediated mechanism of toxicity in the common mussel, Mytilus edulis L., and other molluscs. Funct Ecol 1990; 4: 415-24.

Madara JL. Functional morphology of epithelium of the small intestine. Compr Physiol 2011; 3: 83-115.

Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci 1993; 84: 407-07.

Noah TK, Donahue B, Shroyer NF. Intestinal development and differentiation. Exp Cell Res 2011; 317(19): 2702-10.

Peulen O, Deloyer P, Dandrifosse G. Short-term effects of spermine ingestion on the small intestine: a comparison of suckling and weaned rats. Reprod Nutr Dev 2004; 44(4): 353-64.

Peulen O, Deloyer P, Grandfils C, Loret S, Dandrifosse G. Intestinal maturation

induced by spermine in young animals. Livest Prod Sci 2000; 66(2): 109-20.

Ren W, Yin Y, Liu G, Yu X, Li Y, Yang G, et al. Effect of dietary arginine

supplementation on reproductive performance of mice with porcine circovirus type 2 infection. Amino acids 2012; 42(6): 2089-94.

Rosalki S, Foo AY. Two new methods for separating and quantifying bone and liver alkaline phosphatase isoenzymes in plasma. Clin Chem 1984; 30(7): 1182-86.

Srivastava S, Mishra S, Tripathi RD, Dwivedi S, Gupta DK. Copper-induced

oxidative stress and responses of antioxidants and phytochelatins in Hydrilla verticillata (Lf) Royle. Aquat Toxicol 2006; 80(4): 405-15.

Vardi N, Parlakpinar H, Ozturk F, Ates B, Gul M, Cetin A, et al. Potent protective effect of apricot and P-carotene on methotrexate-induced intestinal oxidative damage in rats. Food Chem Toxicol 2008; 46(9): 3015-22.

Wang J, Li G, Tan B, Xiong X, Kong X, Xiao D, et al. Oral administration of

putrescine and proline during the suckling period improves epithelial restitution after early weaning in piglets. J Anim Sci 2015; 93(4): 1679-88.

Winston GW, Di Giulio RT. Prooxidant and antioxidant mechanisms in aquatic organisms. Aquat Toxicol 1991; 19(2): 137-61.

Yang CC, Fang JY, Hong TL, Wang TC, Zhou YE, Lin TC. Potential antioxidant properties and hepatoprotective effects of an aqueous extract formula derived

420 from three Chinese medicinal herbs against CCl4-induced liver injury in rats. Int

421 Immunopharmacol 2013; 15(1): 106-13.

422 Yin J, Ren W, Liu G, Duan J, Yang G, Wu L, et al. Birth oxidative stress and the

423 development of an antioxidant system in newborn piglets. Free Radic Res 2013;

424 47(12): 1027-35.

425 Zhang W, Chen Q, Mai K, Xu W, Wang X, Liufu Z. Effects of dietary a - lipoic acid

426 on the growth and antioxidative responses of juvenile abalone Haliotis discus

427 hannai Ino. Aquac Res 2010; 41(11): e781-e87.

Table 1 Effects of graded levels of spermine and its time extension on morphology of jejunum in weaned rats.1

Item 3-d treatments 7-d treatments SEM P

C SO.2 SO.4 c SO.2 SO.4 s TS SxTS

Villus height |im 367.24a 373.84a 368.91a 396.95ab 436.27ab 466.91b 11.21 0.345 0.003 0.388

Villus width, |im 108.83 109.75 113.55 85.06 106.02 104.20 3.03 0.174 0.037 0.337

Crypt depth, |im 147.23a 143.99a 146.30a 162.79ab 168.02b 170.12b 3.16 0.891 0.001 0.780

Villus height/crypt depth 2.54 2.63 2.52 2.44 2.61 2.74 0.06 0.639 0.790 0.591

Villus surface area, mm2 0.13 0.13 0.13 0.11 0.15 0.15 0.01 0.116 0.639 0.309

C = control; S0.2 = 0.2 ¡imol spermine/g BW; SO.4 = 0.4 ¡imol spermine/g BW; S =spermine; TS = treatments.

ab Within a row, means with different superscript letters significantly differ (P < 0.05) for comparison between C, SO.2, SO.4.

1 Data are presented as means ± SEM, n = l.

Table 2 Effects of graded levels of spermine and its time extension on morphology of ileum in weaned rats.1

Item 3-d treatments 7-d treatments SEM P

C SO.2 SO.4 C SO.2 SO.4 S TS SxTS

Villus height, |im 247.15 220.78 269.88 237.74 249.99 257.21 5.97 0.138 0.840 0.277

Villus width, |im 83.35 83.53 96.49 78.5 89.81 90.36 2.06 0.051 0.660 0.361

Crypt depth, |im 135.00 127.31 142.86 155.37 132.00 149.14 3.60 0.110 0.140 0.598

Villus height/crypt depth 1.88b 1.73ab 1.89b 1.54a 1.9b 1.73ab 0.04 0.388 0.133 0.017

Villus surface area, mm2 0.06 0.06 0.08 0.06 0.07 0.07 0.00 0.035 0.770 0.178

C = control; S0.2 = 0.2 ¡imol spermine/g BW; SO.4 = 0.4 ¡imol spermine/g BW; S =spermine; TS = treatments.

ab Within a row, means with different superscript letters significantly differ (P < 0.05) for comparison between C, SO.2, SO.4.

1 Data are stated as means ± SEM, n = l.

Table 3 Effects of graded levels of spermine and its duration extension on enzymes activities of jejunum in weaned rats.1

3-d treatments 7-d treatments P

Item _ _SEM _

C SO.2 SO.4 C SO.2 S0.4 S TS SxTS

Protein content, mg/g tissue 124. .88 126. .29 131. .39 126. 34 123. .77 128. .81 2.01 0.569 0.778 0.910

Lactase activity, U/mg protein 8. .42 7. .39 5. .68 10. 59 8. .32 7. .70 0.53 0.091 0.100 0.859

Maltas e activity, U/mg protein 186. .64 172. .06 159. .04 167. 91 140. .20 101. .41 9.03 0.090 0.039 0.633

Sucrase activity, U/mg protein 58. .69 65. .35 63. .51 80. 09 74. .22 63. .14 3.48 0.701 0.168 0.465

Alkaline phosphatase activity, U/g protein 310. 90a 473. ,28ab 503. 07b 572. 15b 554. 09b 596. 60b 23.91 0.197 0.005 0.257

C = control; S0.2 = 0.2 ¡imol spermine/g BW; SO.4 = 0.4 ¡imol spermine/g BW; S =spermine; TS = treatments.

ab Within a row, means with different superscript letters significantly differ (P < 0.05) for comparison between C, SO.2, SO.4.

1 Data are presented as means ± SEM, n = l.

Table 4 Effects of graded levels of spermine and its duration extension on enzymes activities of ileum in weaned rats.1

Item 3-d treatments 7-d treatments SEM P

C SO.2 SO.4 C SO.2 SO.4 S TS SxTS

Protein content, mg/g tissue 91 .60 89.64 87.96 101 .78 98.11 112.63 2.79 0.602 0.009 0.386

Lactase activity, U/mg protein 11 .37 6.74 5.48 5 .04 7.62 6.01 0.86 0.499 0.339 0.163

Maltase activity, U/mg protein 269 .54 233.15 214.8 209 .98 243.83 172.88 14.86 0.379 0.324 0.618

Sucrase activity, U/mg protein 89 .74 71.12 65.96 72 .61 88.63 64.86 6.49 0.578 0.986 0.584

Alkaline phosphatase activity, U/g protein 845 .81 1212.38 1599.93 685 .36 1432.87 826.33 114.75 0.098 0.277 0.180

C = control; S0.2 = 0.2 ¡imol spermine/g BW; SO.4 = 0.4 ¡imol spermine/g BW; S =spermine; TS = treatments. 1 Data are presented as means ± SEM, n = l.

Table 5 Effects of graded levels of spermine and its duration extension on the antioxidant status of jejunum in weaned rats.1

3-d treatments 7 -d treatments p

Item C SO.2 SO.4 C SO.2 SO.4 SEM S TS S x TS

MDA, nmol/mg protein 0.31c 0 ,31c 0. 25b 0. ,26bc 0. 18a 0.17a 0.01 0.001 0.000 0.094

ASA, U/g protein 150.13a 157 54ab 212. 95c 163. 13ab 168. 8?ab 178.16b 4.41 0.000 0.562 0.003

AHR, U/mg protein 170.58a 225 ,22bc 259. 37c 205. llab 201. 19ab 246.80c 6.81 0.000 0.945 0.053

T-SOD, U/mg protein 80.99bc 89 ,63c 97. 60d 71. 59a 72. 97ab 89.81cd 1.96 0.000 0.000 0.307

CAT, U/mg protein 3.57a 5 ,12b 6. 24c 3. 49a 5. 13b 6.22c 0.21 0.000 0.886 0.983

GSH, mg/g protein 4.05a 8 ,99b 9. 84b 8. 88b 9. 41b 10.58b 0.43 0.000 0.000 0.001

T-AOC, U/mg protein 1.58ab 1 ,90cd 2. 08d 1. 50a 1. 69abc 1.81bc 0.05 0.000 0.017 0.536

C = control; S0.2 = 0.2 ¡imol spermine/g BW; SO.4 = 0.4 ¡imol spermine/g BW; S =spermine; TS = treatments; MDA = malondialdehyde; ASA = anti-superoxide anion; ARH = anti-hydroxyl radical; T-SOD = total superoxide dismutase; CAT = catalase; GSH = glutathione; T-AOC = total antioxidant capacity.

ad Within a row, means with different superscript letters significantly differ (P < 0.05) for comparison between C, SO.2, SO.4. 1 Data are presented as means ± SEM, n = l.