Effects of Acute and Chronic Cold Stress on Antioxidant Function in Intestinal Tracts of Chickens

Xu Ming,Yu Xian-yi,Li Jin-long,Han Yan-hui,Li Shu,and Xu Shi-wen

College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China

Introduction

Stress is known as the disruption of homeostasis,which is managed by the complex formations of physiological and behavioural adaptive responses of organisms (Johnson et al.,1992).For animals (especially newborn animals) in cold regions,cold is a main stressor (Hanmin and Shize,1999).Studies have revealed that cold stress could shift the antioxidant balance of the organism in the form of amplification of xanthine oxidase prooxidant enzymatic activity in the brain and liver,and a decrease of myeloperoxidase activity in blood neutrophiles of rats (Ahmad and Mitsuhiro,2009).The cold exposure may be reflected in an elevated metabolic rate and possibly also increased production of reactive oxygen species,e.g.hydrogen peroxide (H2O2),hydroxyl radicals (HO) and superoxide anion radicals (O2•-),which cause lipid peroxidation (Selman et al.,2000;Heise et al.,2003). The main antioxidative enzymes for the detoxification of reactive oxygen species in all organisms are superoxide dismutase (SOD),catalase (CAT) and glutathione peroxidase (GPx).Enzymatic degradation of O2•-to H2O2is ensured by SOD.Monari et al.(2007) reported that the extent of potential damage,which is defined as a disruption of the pro-antioxidant balance by oxidative stress,is dependent on the effectiveness of the antioxidant enzyme superoxide dismutase,and changes in its activity have been proposed as a biomarker of pollutant-mediated oxidative stress.The main source of Malondialdehyde (MDA) in biological samples is the peroxidation of polyunsaturated fatty acid (FA) (Winston et al.,1991).Therefore,we studied different tissues for MDA measurement as an index of oxidative damage in neonatal chickens exposed to lowtemperature.The level of organisms'total antioxidant capacity (T-AOC) has very important significance to comprehensively judge the organisms'body fluid,cell,histaminase system and non-enzymatic system.Therefore,detection the level of the T-AOC has very important significance.

The cold-specific activation of medullary TRH neurons and related vagal activation to the gut provide insight to the differential gastrointestinal tract motor response to cold (stimulatory) compared with other stressors (inhibitory) (Tache et al.,2001).The intestinal tract,as the body's largest organ contacted with the external environment,provides a regulatory barrier to which the animals are exposed to a large assortment of nutrients,microbes and exogenous toxins.The intestine permits the exchange of beneficial nutrients into the systemic circulation,while simultaneously prevents penetration of pathogenic organisms and toxic compounds (Hirata et al.,2007).Thus,maintaining the integrity of the epithelium lining the gastrointestinal tract is of great importance,ensuring its absorptive and protective functions are not compromised (Leon et al.,2005).Because of the unique anatomical features in blood vessels of the intestinal tract,making it more vulnerable to damage under cold stress circumstance but it had not been reported about the mechanism of cold stress on intestinal damage although intestinal tract was an important target organ in cold stress.Studies have shown that rat gastric mucosa was seriously injuried when stimulated with constraint and immersed in 25℃ cold water.Moreover,there were many reports concerned to prenatal and neonatal rodents exposed in cold stress environment at home and abroad,but few studies focused on domestic animals,poultry were fewer.

In this study,we through investigated autopsy change and the content of antioxidation function (T-AOC,SOD and MDA) in duodenum,jejunum and ileum in chickens in hope of that preliminarily revealed the damage mechanisms and the resistant mechanism of cold stress on chickens.It could produce more data for studies of cold stress of chickens in the future.It was to explore methods which could improve animal's coldresist ability to provide a scientific basis and experimental foundation for maintaining animals'normal physiological functions in cold stress environments.

Materials and Methods

Birds and tissue collection

A total of 80 1-day-old male chickens were purchased from Weiwei Co.,Ltd.(Harbin,China),the chickens were maintained in facility under the temperature of (30±2)℃ and were given commercial diet and water.At the age of 15 days,the chickens were randomly divided into 12 groups at average,and cold stress was initiated.The temperature of cold stress was (12±1)℃ and the duration of acute cold stress were 0,1,3,6,12 and 24 h,the duration of chronic cold stress were 5,10 and 20 days.0 h was the control group of acute cold stress,and there were three control groups for chronic cold stress groups,so there were 12 groups in all including four control groups and eight treatment groups (five chickens of each group).Chickens were killed by decapitation after the corresponding duration of cold stress,the duodenum,jejunum and ileum of each chicken were collected,and cut open,washed with cold sterile PBS and observed by macroscopical the change of the duodenum,jejunum and ileum,then immediately frozen on dry ice,and then stored at –80℃ for SOD,MDA,NO,and NOS detection.

Autopsy change of duodenum

The duodenum,jejunum and ileum were taken at each time points.The condition of injury of duodenum mucosa was observed by macroscopical.

Detection of antioxygen function

The chemical colorimetric method was used to detect the changes of the T-AOC,SOD activities and MDA contents in duodenum,jejunum and ileum.

The duodenum,jejunum and ileum were taken at each time points those were homogenized in physiological saline,centrifuged at 700 g and supernatant was collected.The T-AOC in the homogenate was determined by chemical colorimetric method (Nanjing Jiancheng Bioengineering Institute,Nanjing,China).SOD activity in the homogenate was assayed by the inhibition,at 25℃,of pyrogallol autoxidation by SOD (with and without sample) and was followed kinetically at 550 nm (Nanjing Jiancheng Bioengineering Institute,Nanjing,China).MDA contents in the homogenate were determined by 2-thiobarbituric acid reactive substance (TBARS) chromometry (Nanjing Jiancheng Bioengineering Institute,Nanjing,China).

Statistical analysis

Statistical analysis of all data was performed using SPSS procedures (version 13;SPSS Inc.Chicago,IL,USA).The effect of cold stress on contents of T-AOC,MDA and SOD in chickens was assessed by 1-way ANOVA and a significant value (P＜0.05) was considered as significant difference.All data showed a normal distribution and passed equal variance testing.Differences between means were assessed using Tukey's honestly significant difference test for post hoc multiple comparisons.Data were expressed as the mean±standard deviation (SD).

Results

Autopsy change of duodenum,jejunum and ileum

Autopsy showed no pathological change in duodenum of control group.The exposure to cold stress produced edema and epithelial damage.During the acute cold stress,there were slightly haemorrhage and hyperemia in duodenum.During the chronic cold stress,there were developments of hemorrhagic and ulcer lesions in duodenum,duodenal wall thickening,in the serosa and mucosal surface we could see bleeding spots,ecchymosis or diffuse hemorrhage.Further,there were round or oval yellow necrosis and deep muscle ulceration in intestinal tract of some chickens,and obvious boundaries with the surrounding organizational.It was in a time-dependent manner,which was the most serious in 20 days group.

Autopsy showed no pathological changes in jejunum and ileum of control group.The exposure to cold stress produced edema and epithelial damage.During the acute cold stress,there were slightly hyperemia in jejunum and ileum.During the chronic cold stress,intestinal tract was swelling and inelastic and there were small bleeding spots on the bowel wall.There were some scattered ulcers as date-stone in jejunum mucosa and ileum mucosa of some chickens.While ulcers were deep to the muscle,overlie a layer of pseudomembranous.It was in a time-dependent manner,which was the most serious in 20 days group.

Effects of acute cold stress on contents of the T-AOC,MDA and SOD in duodenum,jejunum and ileum of chickens

Effects of acute cold stress on the T-AOC,SOD activities and MDA contents in duodenum are presented in Table 1.Compared with the control group,the T-AOC and SOD activities in duodenum,1,3,6,and 12 h treatment groups significantly (P＜0.05) increased,but 24 h treatment group decreased control value (P＞0.05).The MDA contents in duodenum 1,3,6,12,and 24 h significantly (P＜0.05) increased.

Effects of acute cold stress on the T-AOC,SOD activities and MDA contents in jejunum are presented in Table 2.Compared with the control group,except 1 h treatment group increased control value (P＞0.05),the T-AOC in jejunum,3,6,12,and 24 h significantly (P＜0.05) increased;SOD activity and MDA content in jejunum,1,3,6,12,and 24 h treatment groups significantly (P＜0.05) increased.

Effects of acute cold stress on the T-AOC,SOD activities and MDA contents in ileum are presented in Table 3.Compared with the control group,the T-AOC in ileum,1 h treatment group decreased control value (P＞0.05),and 12 and 24 h treatment groups significantly (P＜0.05) decreased,while 3 and 6 h significantly (P＜0.05) increased,MDA content in ileum,1,3,6,12,and 24 h treatment groups significantly (P＜0.05) increased.SOD activity in ileum,1 h treatment group serum but 3,6,12,and 24 h treatment groups significantly (P＜0.05) decreased.

Table1 Effects of acute cold stress on contents of T-AOC,MDA and SOD in duodenum

Table2 Effects of acute cold stress on contents of T-AOC,MDA and SOD in jejunum

Table3 Effects of acute cold stress on contents of T-AOC,MDA and SOD in ileum

Effects of chronic cold stress on contents of T-AOC,MDA and SOD in duodenum,jejunum and ileum of chickens

Effects of chronic cold stress on the T-AOC,SOD activities and MDA contents in duodenum are presented in Table 4.Compared with the corresponding control groups,chronic cold stress resulted in a significant decrease (P＜0.05) of the T-AOC in duodenum and a significant (P＜0.05) increase of the MDA contents in duodenum.The SOD activity,in duodenum,5 and 10 days significantly decreased (P＜0.05),but 20 days significantly (P＜0.05) increased.

Effects of chronic cold stress on the T-AOC,SOD activities and MDA contents in jejunum are presented in Table 5.Compared with the corresponding control groups,chronic cold stress resulted in a significant decrease (P＜0.05) of the T-AOC and SOD activities in jejunum and a significant (P＜0.05) increase of the MDA contents in jejunum,but 20 days treatment group of the MDA contents increased corresponding control value (P＞0.05).

Effects of chronic cold stress on the T-AOC,SOD activities and MDA contents in ileum are presented in Table 6.Compared with the corresponding control groups,chronic cold stress resulted in a significant decrease (P＜0.05) of the T-AOC and SOD activities in ileum but 20 days treatment group of the T-AOC contents decreased corresponding control value (P＞0.05),and a significant (P＜0.05) increase of the MDA contents in ileum.

Table4 Effects of chronic cold stress on contents of T-AOC,MDA and SOD in duodenum

Table5 Effects of chronic cold stress on contents of T-AOC,MDA and SOD in jejunum

Table6 Effects of chronic cold stress on contents of T-AOC,MDA and SOD in ileum

Discussion

Stress is a series of responses by which animals protect themselves against suboptimal environment conditions,the environment conditions are stress factors and there are many stress factors in nature.Cold has many effects on animals and an increasing number of studies have shown that cold stress may alert the antioxidant protection system of the organism (Shustanova et al.,2004;Gümüşlü et al.,2002).Cold stress,which is characterized by the increase inmetabolic rate,leads to alterations in some antioxidant enzyme activities.These alterations are explained as a mechanism resisting the negative effects of reactive oxygen species (Selman et al.,2000).Several studies have investigated whether exposure to low temperature results in compensatory changes taking place in the antioxidant defence system (Barja et al.,1991;Ohno et al.,1991;Bondarenko et al.,1999),although there has been no detailed and comparable study investigating the effects of cold stress on protein modification and lipid peroxidation in different tissues.

Studies have shown that the gastrointestinal tract occurred ischemia and hypoxia,when stressed (Secchi et al.,2008).Moreover,as a special intestinal vascular anatomy and characteristics of flow oxygen exchange mechanism,it was more likely to occur hypoxicischemic damage under stress,making the intestinal epithelial edema,breaking the connection epithelial cell membrane and cell,cell necrosis,epithelium from the top of villi even the full-thickness began to fall off formation of mucosal ulceration,and leading to increased intestinal permeability and thus bacterial and endotoxin translocation (Boros et al.,1995;Pedersen et al.,2000;Rowell,1983).In the same time,ischemia and hypoxia will lead to local produces a large number of acidic metabolites,acidosis itself can directly cause block of cell metabolism,tissue lesion,and indirectly worsene tissue edema by increasing extracellular calcium influx,causing epithelial permeability increased.Besides,intestinal cells will produce a large number of toxic reactive oxygen metabolites under hypoxic conditions,containing hydrogen peroxide (H2O2),hydroxyl radicals (HO•) and superoxide anion radicals (O2•-) and so on,further damage to the intestinal mucosa.In addition,intestinal mucosal cells subjected to hypoxia stress injury more significantly because there is rich of xanthine oxidase in intestine tissue (Mitsuoka et al.,2000).Observation on the phenomenon of gastrointestinal mucosa congestion suggested that the cold stress can lead to peptic ulcer disease.

SOD reflects the production of radical and MDA reflects the degree of lipid peroxidation (Ilhan et al.,2001).SOD can resist oxygen free radical,promote the regulating function of H2O2density,protect tissues and cells by catalyzing the O2•-dismutation reaction.Considering SOD activity as an indirect measure of O2•-production,they also determine the positive relationship between tissue-specific SOD activity and index of superoxide production.Radical theory considered that the strength of organism defense system's antioxidant capacity had close relation to healthy level of body.In our study,at the beginning of acute cold stress,there was an significant increase (P＜0.05) of SOD activity in duodenum,jejunum and ileum and then there was an significant decrease (P＜0.05) of SOD activity in duodenum,jejunum and ileum.Kaushik and Kaur (2003) found that chronic exposure to cold stress increased catalase activity,as in our study,although it decreased total SOD in duodenum,jejunum and ileum.

The defense system has enzyme promotion system and non-enzyme promotion system.Mainly through three ways to complete its protective oxidation: elimination of free radicals and reactive oxygen species in avoid to lipid peroxidation;decomposition of peroxides,block peroxide chain;remove catalytic metal ions,while defense system build synergistic effect,and the compensatory and dependence role between the various components.This function degrades often lead to various diseases,thus determination of the T-AOC has very important significance.In this study,we found that the T-AOC of the body rapidly and significantly (P＜0.05) increased during acute cold stress.It noted that body had strong regulated capability to the rapid removal of the body produced during the cold stress on the animal's own growth adversely and a variety of free radicals.

In this study,we found a significant increase in brain and heart MDA levels of low-temperature-exposed neonatal chickens compared to those of control chickens.It indicated the existence of some oxidative stresses in brain and heart of low-temperature-exposed chickens (Bondarenko et al.,1999).Moreover,a previous work demonstrated that an increased MDA concentration further attenuated the activity of enzymes involved in the detoxification of hydrogen peroxide,hydroxyl,and superoxide radicals (Patel et al.,2006).In our study,during acute cold stress and chronic cold stress,the MDA contents significantly (P＜0.05) increased in duodenum,jejunum and ileum of chickens.It showed higher duodenum,jejunum and ileum MDA levels in low-temperature exposed neonatal chickens that might be the consequence of modulation in oxidative metabolism and/or higher oxidative stress.High MDA levels in duodenum,jejunum and ileum suggested that the lipids were heavily peroxidized in these tissues of low-temperature-exposed chickens that presumably associated with increased free radicals or diminished antioxidant capacity: lower levels of antioxidants,modulation in antioxidant defense system enzymes or their activities and/or their susceptibilities to oxidants.

Conclusions

This study showed that both acute and chronic cold stresses could cause duodenum,jejunum and ileum oxidative stress,change T-AOC,MDA contents and SOD activity,which were related to the intestinal damage process.However,the intestinal damage and oxidative stress mechanism,with aspect to the T-AOC,MDA contents and SOD activity may be more important and requires more studies.

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