7
RESEARCH Oxidative stress in farmed minks with self-biting behavior Defa Sun a , Jianhua Wang b , Xiurong Xu a a Research Group of Animal Biochemistry, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, People’s Republic of China; and b Research Group of Clinical Veterinary Medicine, College of Veterinary Science, Northwest A&F University, Yangling, Shaanxi, People’s Republic of China. KEYWORDS: mink; self-biting behavior; oxidative stress; malondialdehyde; protein carbonyl Abstract Self-biting behavior (SBB) is a serious behavioral disorder in farmed minks, but little is known about the biological basis of this disorder. The present study examined for the first time the he- patic and cerebral oxidative stress biomarker levels in SBB minks and compared them with those in normal ones to study the association of oxidative stress and SBB in minks. Calcium-activated adeno- sine triphosphatase (Ca 21 ATPase) activities were also determined to investigate whether the Ca 21 pump in brain is affected. Twenty male SBB minks and 20 male normal minks were chosen in Decem- ber. The brain and liver of each mink were harvested immediately after slaughter to test oxidative stress biomarkers. All parameters were determined by using a spectrophotometer. Our findings were as fol- lows: SBB minks produced more malondialdehyde with 51% increase in brain (P , 0.01) and 22% increase in liver (P , 0.01) compared with normal minks. Similarly, the hepatic and cerebral protein carbonyls in SBB minks were 18.5% (P , 0.01) and 30% higher (P , 0.001), respectively. Glutathione peroxidase activities in brain and catalase activities in both tissues of SBB minks were markedly lower than those of normal group (P , 0.01). However, SBB minks had higher total superoxide dismutase (SOD), Cu/Zn-SOD, and Mn-SOD activities in both tissues (P , 0.05 or P , 0.01). Significant deple- tion of glutathione and vitamin E levels were observed in SBB minks (P , 0.01 or P ,0 .05). Calcium- activated adenosine triphosphatase activities in the brain of SBB minks were inhibited (P , 0.01). Our results supported the oxidative stress hypothesis in minks with SBB. Ó 2013 Elsevier Inc. All rights reserved. Introduction Patients with neurodegenerative diseases, including Lesch– Nyhan syndrome (Cauwels and Martens, 2005), Cornelia de Lange syndrome (Hyman et al., 2002), Rett syndrome (Sansom et al., 1993), Tourette syndrome (Mathews et al., 2004), and autism (Baghdadli et al., 2003; Chauhan and Chauhan, 2006), often have self-injurious behavior (SIB). Some animals, such as farmed mink (Bildsøe et al., 1990; Mason, 1993, 1994) and housed monkeys (Kraemer et al., 1990, 1997), also have SIB. Because minks with SIB injure themselves by biting their body, especially their tail, SIB in mink is also named as tail-biting behavior. However, tail- biting can be self-performed or performed by another mink; furthermore, some minks bite other parts of their body, instead of tail. Therefore, we called this behavior self-biting behavior (SBB). A mink with SBB bites its tail or rear quarter suddenly, performs this behavior for 30 seconds to 2 minutes each time and repeats the behavior several times each day. In China, reported incidence of self- biting in farmed minks ranges from 5% to 20%. Address for reprint requests and correspondence: Xiurong Xu, PhD in Animal Genetics, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, People’s Republic of China; Tel: +8615-09128-4067; Fax: 18629-8709-1973. E-mail: [email protected] 1558-7878/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. doi:10.1016/j.jveb.2012.01.002 Journal of Veterinary Behavior (2013) 8, 51-57

Oxidative stress in farmed minks with self-biting behavior

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Page 1: Oxidative stress in farmed minks with self-biting behavior

Address for

Animal Genetic

A&F University

Tel: +8615-0912

E-mail: xiur

1558-7878/$ - s

doi:10.1016/j.jv

Journal of Veterinary Behavior (2013) 8, 51-57

RESEARCH

Oxidative stress in farmed minks with self-biting behavior

Defa Suna, Jianhua Wangb, Xiurong Xua

aResearch Group of Animal Biochemistry, College of Animal Science and Technology, Northwest A&F University,Yangling, Shaanxi, People’s Republic of China; andbResearch Group of Clinical Veterinary Medicine, College of Veterinary Science, Northwest A&F University, Yangling,Shaanxi, People’s Republic of China.

KEYWORDS: Abstract Self-biting behavior (SBB) is a serious behavioral disorder in farmed minks, but little is

mink;self-biting behavior;oxidative stress;malondialdehyde;

protein carbonyl

known about the biological basis of this disorder. The present study examined for the first time the he-patic and cerebral oxidative stress biomarker levels in SBB minks and compared them with those innormal ones to study the association of oxidative stress and SBB in minks. Calcium-activated adeno-sine triphosphatase (Ca21ATPase) activities were also determined to investigate whether the Ca21

pump in brain is affected. Twenty male SBB minks and 20 male normal minks were chosen in Decem-ber. The brain and liver of each mink were harvested immediately after slaughter to test oxidative stressbiomarkers. All parameters were determined by using a spectrophotometer. Our findings were as fol-lows: SBB minks produced more malondialdehyde with 51% increase in brain (P , 0.01) and 22%increase in liver (P , 0.01) compared with normal minks. Similarly, the hepatic and cerebral proteincarbonyls in SBB minks were 18.5% (P, 0.01) and 30% higher (P, 0.001), respectively. Glutathioneperoxidase activities in brain and catalase activities in both tissues of SBB minks were markedly lowerthan those of normal group (P , 0.01). However, SBB minks had higher total superoxide dismutase(SOD), Cu/Zn-SOD, and Mn-SOD activities in both tissues (P , 0.05 or P , 0.01). Significant deple-tion of glutathione and vitamin E levels were observed in SBB minks (P, 0.01 or P,0 .05). Calcium-activated adenosine triphosphatase activities in the brain of SBB minks were inhibited (P , 0.01). Ourresults supported the oxidative stress hypothesis in minks with SBB.� 2013 Elsevier Inc. All rights reserved.

Introduction

Patients with neurodegenerative diseases, including Lesch–Nyhan syndrome (Cauwels and Martens, 2005), Corneliade Lange syndrome (Hyman et al., 2002), Rett syndrome(Sansom et al., 1993), Tourette syndrome (Mathews et al.,2004), and autism (Baghdadli et al., 2003; Chauhan andChauhan, 2006), often have self-injurious behavior (SIB).

reprint requests and correspondence: Xiurong Xu, PhD in

s, College of Animal Science and Technology, Northwest

, Yangling, Shaanxi 712100, People’s Republic of China;

8-4067; Fax: 18629-8709-1973.

[email protected]

ee front matter � 2013 Elsevier Inc. All rights reserved.

eb.2012.01.002

Some animals, such as farmed mink (Bildsøe et al., 1990;Mason, 1993, 1994) and housed monkeys (Kraemer et al.,1990, 1997), also have SIB. Because minks with SIB injurethemselves by biting their body, especially their tail, SIB inmink is also named as tail-biting behavior. However, tail-biting can be self-performed or performed by anothermink; furthermore, some minks bite other parts of theirbody, instead of tail. Therefore, we called this behaviorself-biting behavior (SBB). A mink with SBB bites itstail or rear quarter suddenly, performs this behavior for30 seconds to 2 minutes each time and repeats the behaviorseveral times each day. In China, reported incidence of self-biting in farmed minks ranges from 5% to 20%.

Page 2: Oxidative stress in farmed minks with self-biting behavior

Figure 1 Picture of the rear of mink with obvious self-biting be-havior and visible wound on the tail.

52 Journal of Veterinary Behavior, Vol 8, No 1, January/February 2013

Furthermore, serious self-biters can proceed to fatal hemor-rhage (Cynthia and Kahn, 2005), tissue damage, and infec-tion (SCAHAW, 2001).

Up to now, very little is known about the biological basisof SBB in minks. It is generally believed this behavioraldisorder is caused by captivity and issues of animal welfare(Mason, 1994, 1996; Jeppesen et al., 2000; Vinke et al.,2002, 2004, 2005). The factors that are often described asbeing relevant for the welfare of mink are as follow: ageat weaning, management systems, and type or presence ofnest boxes and bedding. For example, minks removedfrom their mothers at 7 weeks of age developed moreself-biting than minks left with their mothers until 6 months(Mason, 1994). There were few studies on biological mech-anism of self-biting induced by bad animal welfare inminks.

There is growing evidence to suggest an associationbetween SIB in animal models or human neurodegenerativediseases and oxidative stress (Gotz et al., 1990; Fahn andCoheb, 1992; Visser et al., 2002; Suzuki et al., 2005;Mori et al., 2007; Wong-Ekkabut et al., 2007). Oxidativestress can cause excessive generation of free radicals andlead to cell damage, membrane permeability increase,and consequent influx of Ca21 in brain, which could resultin mental disturbances that may constitute a backgroundfor SIB.

Several defense mechanisms are evolved in decreasingthe degree of free radicals (Cheeseman and Slater, 1993).These defense mechanisms include the antioxidant en-zymes, such as glutathione peroxidase (GPx), catalase(CAT), and superoxide dismutase (SOD), as well as the non-enzymatic antioxidants, such as glutathione (GSH) and vita-min E (VE).

To our knowledge, there were no available data onoxidative stress in minks with SBB. In the present study, weinvestigated antioxidant enzymes (GPx, CAT, and SOD)activities and the levels of 2 free radical scavengers (GSHand VE), malondialdehyde (MDA, marker of lipid perox-idation [LP]), and protein carbonyls (PC, an index ofprotein oxidation) in liver and brain of minks with SBB andnormal minks to reveal whether SBB in minks has anyassociation with oxidative stress.

Calcium-activated adenosine triphosphatases (Ca21-ATPases) are adenosine triphosphate (ATP)-dependentCa21 pumps that transport Ca21 against its concentrationgradient into the endoplasmic reticulum (ER) lumen orout of the cell. It was reported that oxidants such as super-oxide (Grover and Samson, 1988) and hydrogen peroxide(H2O2) (Grover et al., 1992) can effectively inhibit Ca21

backflow by inhibiting both ER Ca21-ATPases andplasma membrane ATPases (Rohn et al., 1993). To investi-gate whether the Ca21 pump in brain cells of SSB minksis affected, Ca21-ATPase activity in brain was alsodetermined.

Materials and methods

Animals

Twenty male SBB minks (minks with obvious SBB andbiting-caused visible wound on body; see Figure 1) and20 male normal minks (minks without SBB and no visiblewound on body) were chosen before pelting in Decemberfrom Dalian Jinzhou Mink Farm in Liaoning Province ofChina. All the selected minks had no wound when the ex-periment started; the wound emerged after mink performedSBB, and the wound site was the biting site. The minkswere observed for 1 day after the wound emerged. For pre-venting unexpected effect of wound treatment on the phys-iology of the self-biting mink, we did not treat the woundduring this 1 day of observation. Considering both animalwelfare and the effect that may be caused by wound infec-tion, we did not observe the selected mink for more than1 day after the wound emerged. All the minks ranged inage from 5 to 6 months. Each mink (Mustela vison) wassingly housed from weaning to pelting and fed the samediet. There was a gap between every 2 neighboring minkcages in the Dalian Jinzhou Mink Farm, thus a mink housedat this farm cannot be bitten by neighboring minks.

Sample collection and preparation

The brain and liver of each mink were harvestedimmediately after slaughter and quickly rinsed in ice-coldphysiological saline, and 1 contralateral hemisphere andpart of the liver from each mink were frozen in liquidnitrogen and stored at 280 �C until use in the followingdays. Approximately 500 mg of the cooled tissue of eachsample was weighed and diluted 10% (w/v) in 20 mM ice-cold Tris–HCl, pH 7.4, homogenized with a homogenizator,and centrifuged at 3,000 ! g for 30 minutes at 4 �C fortesting enzyme activities and protein, GSH, MDA, andPC levels in the supernatant fraction. A total of 500 mg

Page 3: Oxidative stress in farmed minks with self-biting behavior

Table 1 GSH and VE levels in the brain and liver of normaland SBB minks

Measurement Tissues Normal mink SBB mink P value

GSH (mg/gprotein)

Liver 29.50 6 5.85 23.05 6 6.22 0.002a

Brain 23.09 6 4.98 17.47 6 4.38 0.001a

VE (mg/gtissue)

Liver 15.10 6 4.45 11.70 6 4.10 0.019b

Brain 10.39 6 2.41 8.19 6 2.07 0.004a

SSB, self-biting behavior.

Note: All of the values are mean 6 SD, and n 5 20 in each group.aValues in the same line are markedly different P , 0.01.bValues in the same line are statistically different P , 0.05.

Sun et al Oxidative stress in SBB minks 53

of the cooled tissue of each sample was homogenized be-fore extraction into xylene for measuring VE concentration.

Determination of protein concentration

The hepatic and cerebral protein concentrations weremeasured by spectrophotometry at 280 nm using bovineserum albumin as standard (Lowry et al., 1951).

Measurement of GSH levels

The hepatic and cerebral GSH concentrations wereestimated according to method by Beutler et al. (1963). Tis-sue homogenate was first deproteinized and then centri-fuged at 2,000 ! g for 10 minutes. After addition ofdithiobisnitrobenzoate and phosphate buffer (pH 8.0) intothe clear supernatants of the samples, the color that devel-oped was read at 412 nm. The concentration of GSH inliver and brain was expressed as milligram of GSH pergram protein of wet tissue.

Measurement of VE levels

The method of VE measurement is based on theoxidation of xylene-extracted tocopherols of the sampleby ferric chloride, and the pink complex of ferrous ionswith bathophenanthroline was measured colorimetrically at536 nm (Desia and Machlin, 1985).

Determination of enzymatic activities of GPx,CAT, and SOD

GPx activity was determined by the method of Pagliaand Valentine (1967). Briefly, GPx catalyzed part of GSHto oxidized GSH, induced by H2O2. The remaining GSHin the solution will react with dithiobis nitrobenzoic acidand produce a yellow substance whose absorbance can bemeasured at 412 nm. One unit of GPx activity was definedas the amount of the enzyme that lowered the concentrationof GSH by 1 mM/min at 37 �C in 1 mg of tissue protein.

CAT activity was tested by the method of Aebi et al.(1987) using H2O2 as substrate. The disappearance ofH2O2 was estimated at 240 nm. Enzyme activity was ex-pressed as units per gram of wet tissue.

Total SOD activity was determined by the methoddescribed by Sun et al. (1988). This method is based onthe principle of inhibition of nitroblue tetrazolium reduc-tion by the xanthine-xanthine oxidase system, which actsas a superoxide generator. One unit of SOD was definedas the enzyme activity causing 50% inhibition in the nitro-blue tetrazolium reduction rate. The amount of Mn-SODactivity was determined by addition of 0.1 M NaCN to in-hibit Cu/Zn-SOD. Cu/Zn-SOD activity was obtained bysubtracting the Mn-SOD activity from the total SOD activ-ity, which was expressed as units per milligram protein ofwet tissue.

Determination of MDA and PC levels

LP in the liver and brain was estimated by calculation ofMDA concentration, which was measured by the thiobar-bituric acid reactive substances assays. This method wasused to obtain a spectrophotometric measurement of thecolor produced during the reaction to thiobarbituric acidwith MDA at 535 nm (Buege and Aust, 1978), which wasexpressed as nanomoles of MDA per milligram protein ofwet tissue.

Tissue PC levels were determined by the spectro-photometric measurement of the formation of 2,4-dinitrophenylhydrazine derivatives. The absorbance wasrecorded at 360 nm, and PC levels were expressed asnanomoles of carbonyl per milligram protein of wet tissue(Levine et al., 1990).

Measurement of Ca21-ATPase activity

The Ca21-ATPase activity was determined by measuringthe quantity of inorganic phosphate liberated from thehydrolysis of ATP according to the method described byWang et al. (1993), and expressed in nanomoles of inor-ganic phosphate per milligram protein per hour.

Statistical analysis

Independent samples t test was performed to evaluategroup differences between the normal group and the SBBgroup. SPSS 13.0 software (SPSS Inc, Chicago, IL) wasused for the statistical analysis. All values were reportedas mean6 SD. For all statistical tests, the 0.05 level of con-fidence was accepted for statistical significance.

Results

GSH and VE levels in liver and brain

GSH is the most abundant nonprotein thiol in the celland a key cellular antioxidant. A significant depletion ofGSH levels was observed both in brain and liver of SBBminks compared with the normal minks (P , 0.01). For

Page 4: Oxidative stress in farmed minks with self-biting behavior

Table 2 Antioxidant enzyme activities in the brain and liver of normal and SBB minks

Measurement Tissues Normal mink SBB mink P value

Total SOD (U/mg protein) Liver 27.23 6 4.46 31.14 6 4.91 0.012a

Brain 18.09 6 25.30 20.87 6 2.29 0.001b

Mn-SOD (U/mg protein) Liver 5.55 6 1.80 7.02 6 2.04 0.02a

Brain 4.71 6 1.05 5.36 6 1.01 0.053Cu/Zn-SOD (U/mg protein) Liver 21.69 6 3.52 24.12 6 3.81 0.042a

Brain 13.49 6 2.61 15.51 6 2.49 0.014a

GPx (U/mg protein) Liver 80.73 6 19.73 71.36 6 14.67 0.096Brain 16.88 6 3.81 13.39 6 3.23 0.005a

CAT (U/g protein) Liver 321.77 6 54.70 212.92 6 52.03 0.000c

Brain 16.87 6 3.81 13.38 6 3.23 0.003b

Note: All of the values are mean 6 SD, and n 5 20 in each group.aValues in the same line are statistically different P , 0.05.bValues in the same line are markedly different P , 0.01.cValues in the same line are significantly different P , 0.001.

54 Journal of Veterinary Behavior, Vol 8, No 1, January/February 2013

example, the GSH level in brain of SBB minks was only76% that of normal minks (Table 1).

As shown in Table 1, VE levels declined both in liver andbrain when minks were in a state of SBB. In all, 22.5% and21.1% of VE decrease was observed in liver and brain,respectively.

SOD activities in liver and brain

To examine the relationship between SBB and SODenzyme activities, total SOD, Cu/Zn-SOD, and Mn-SODactivities in the liver and brain were measured. TotalSOD and Cu/Zn-SOD activities in both tissues andMn-SOD activities in liver of SBB minks were markedlyor significantly higher than those in liver of normalminks (P , 0.05 or P , 0.01). For example, total SODactivities in liver and brain of SBB minks were 14.4%and 15.5% higher, respectively, than those of normalminks (Table 2).

GPx and CAT activities in liver and brain

GPx and CAT enzyme activities in liver and brain of the2 groups are shown in Table 2. In comparison with those of

Table 3 MDA and PC levels in the brain and liver of normaland SBB minks

Measurement Tissues Normal mink SBB mink P value

MDA (nmol/mgprotein)

Liver 1.40 6 0.28 1.71 6 0.40 0.007a

Brain 1.67 6 0.45 2.54 6 0.69 0.000b

PC(nmol/mgprotein)

Liver 3.14 6 0.90 3.72 6 0.89 0.05c

Brain 3.37 6 0.50 4.51 6 1.03 0.000b

Note: All of the values are mean 6 SD, and n 5 20 in each group.aValues are different when P , 0.01.bValues are different when P,0.001.cValues are different when P , 0.05.

the animals in the normal group, CAT activities in bothorgans of SBB minks sharply reduced (P , 0.01), with a34% decrease in liver and 21% decrease in brain. GPxactivities in both tissues of SBB minks were also inhibited,but only in brain, they were statistically lower than those ofnormal minks.

MDA and PC levels in liver and brain

The LP, as an indication of the destruction of cellularmembrane structures, and measured as the formation ofMDA, showed significant differences, with a 51% increasein brain and 22% increase in liver of minks with SBB whencompared with minks in normal condition (Table 3).

Oxidative stress can alter proteins in many ways. Thecommon method to assess such alterations is to measure thecarbonyl groups of the oxidized proteins. As shown inTable 3, minks with SBB showed a statistically significantelevation of the PC content, with a 30% increase in brainand 18.5% increase in liver compared with the normal ones.

ATPase activities in brain

Ca21-ATPases are ATP-dependent Ca21 pumps thattransport Ca21 against its concentration gradient into theER lumen or out of the cell. As shown in Table 4, Ca21-ATPase activities in brain were inhibited when minkswere in a state of SBB (P , 0.05).

Table 4 Difference of cerebral Ca21-ATPase activitiesbetween normal and SBB minks

Measurement Tissues Normal mink SBB mink P value

ATPase (nmol/mgprotein)

Brain 5.51 6 1.14 4.29 6 0.81 0.024a

aValues are different when P , 0.05.

Page 5: Oxidative stress in farmed minks with self-biting behavior

Figure 2 Differences in GSH, VE, MDA, and PC concentrationsbetween liver and brain of minks with self-biting behavior. Dataare the mean values 6 standard error for 20 minks. Unit forGSH, VE, MDA, and PC is mg/g protein, mg/g tissue, nmol/mgprotein, and nmol/mg protein, respectively. Statistically significantdifferences between liver and brain are indicated by ***P, 0.001.MDA, malondialdehyde; GSH, glutathione; VE, vitamin E; PC,protein carbonyls.

Sun et al Oxidative stress in SBB minks 55

Difference in degree of oxidative stress in brainand liver

Difference in degree of oxidative stress in brain and liverwas analyzed. As shown in Table 2, enzyme activities of the3 measured antioxidant enzymes in minks’ liver weresharply higher than those in brain (P , 0.001). For exam-ple, CAT activity in liver is 16-fold of that in brain for SBBminks (Table 2). The liver also contains much higher levelsof GSH and VE than brain (Figure 2). Accumulations ofboth MDA and PC were much more apparent in brainthan in liver, especially when minks were in a state ofSBB (Figure 2).

Discussion

Studies in recent years have proved that oxidative stress,which can lead to free radical-mediated cell damage andneuronal dysfunction, is associated with SIB (Mori et al.,2007; Wong-Ekkabut et al., 2007; Gu et al., 2008; Kitaet al., 2009). Free radicals are highly reactive and capableof damaging many biological macromolecules such aslipids and protein and can result in accumulation of oxida-tively modified lipids and proteins (Pacifici and Davies,

1991). Our present study also provided evidences support-ing the role of oxidative stress in the pathophysiology ofSBB in minks. The support came from the observation ofsharply elevated MDA and PC contents in the brain andliver of SBB minks. Histological damage has been ob-served in cells of the cerebrum, cerebellum, and medullaoblongata of minks with SBB (Sun et al., 2010). This dam-age might be caused by the observed intense oxidativestress in mink.

GSH is the most prevalent low-molecular-weight anti-oxidant in animal cells and very important in maintainingintracellular reduction–oxidation equilibrium, and protectsprotein and enzyme molecules from endogenous oxidiza-tion. Observed depletion of GSH has been reported in manystudies on neurodegenerative diseases such as autism(Geier et al., 2009; Vojdani et al., 2008) and Parkinson dis-ease (Sofic et al.,1992; Jha et al., 2000) in humans or in-duced oxidative stress in animal models (Zaidi and Banu,2004; Nemmiche et al., 2007). In the current study, animalswith SBB showed significant decrease in both hepatic andcerebral reduced GSH levels compared with the normalones. Inhibition of cysteine transport (Cho and Bannai,1990), deficiency of sulfur amino acid in diet, or increasedoxidative stress all can lead to decline of reduced GSH in tis-sues. It is worth noting that the incidence of SBB is more fre-quent during hair growing than other seasons in mink farms.Animal hair is rich in cysteine, which is 1 of the 3 substratesin GSH synthesis. Further study should be performed to in-vestigate whether the competition for cysteine with hairgrowing contributes to the decline of GSH level.

VE is the most important lipid-soluble antioxidant. Itprotects membranes from oxidation by reacting with lipidradicals produced in the LP chain reaction (Traber andAtkinson, 2007). Depletion of reduced VE in present studyprovided another evidence for supporting that SBB inminks is associated with oxidative stress.

Antioxidant enzymes can prevent biological macromol-ecules from oxidative damage. Disruption of antioxidantdefense mechanisms in SBB minks was manifested by theinhibition of GPx and CAT activities. However, contrary tothe observed decrease in many studies on neurodegenera-tive diseases (Thome et al., 1997; Marcus et al., 1998;Johannesson et al., 2003; Schuessel et al., 2005; Casadoet al., 2008; Boll et al., 2008), SOD activities, including to-tal SOD, Cu/Zn-SOD in mitochondria, and Mn-SOD incytosol, elevated in tissues of SBB minks. Several investi-gators have reported accordant results in some studies onautistic (Al-Gadani et al., 2009) and schizophrenic patients(Ookawara et al., 2000; Kuloglu et al., 2002a; Kunz et al.,2008; Zhang et al., 2009). SOD can convert O2

2 to H2O2,which is degraded subsequently by GPx and CAT to water.

Increased SOD activities together with significant inhi-bition of GPx and CAT and depletion of GSH and VE couldresult in H2O2 stress and may contribute to the accumula-tion of MDA and PC investigated in SBB minks. As the pri-mary antioxidant defense system against superoxide

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56 Journal of Veterinary Behavior, Vol 8, No 1, January/February 2013

radicals and 1 of the oxidative stress response proteins(Verhasselt, 1998; Rivollier et al., 2006), SOD may expresshigh level in response to intense superoxide radical envi-ronment in SSB minks.

As an adaptive response, increase in CAT or GPxactivity was also observed in studies on induced oxidativestress (Jarrett and Boulton, 2005; Garrel, 2007) or humandiseases (Kuloglu et al., 2002b), but not found in presentstudy.

Our results demonstrated the enzyme activities of GPxand CAT, and the concentration of GSH and VE both inliver and brain markedly decreased in SBB minks com-pared with the normal minks, but no visible histologicalchanges in liver of SBB minks were observed (Sun,unpublished observation). As shown in present study, theliver has significantly higher antioxidant enzyme activitiesand higher nonenzymatic free radical scavenger levels thanthe brain. Therefore, it is not as vulnerable to oxidativestress as brain.

SBB in minks is a behavior associated with intermittentexcitation. Excitation behavior in animals is closely relatedwith Ca21 influx from extracellular flow to intracellular,transported by L-type Ca21 channel, and Ca21 backflowto extracellular after excitation, transported by Ca21-ATPase. Abnormal Ca21 influx or backflow will lead to ab-normal excitation. L-type Ca21 channel agonist (6)-Bay K8644 could result in abnormal Ca21 influx and provokeSBB in animal model (Kasim et al., 2002). The decreaseof Ca21-ATPase activities in brain of SBB minks indicatedinhibition of Ca21 backflow, which may contribute to Ca21

retention in cytoplasm, and subsequent abnormal responsesuch as intermittent excitation.

Conclusion

In summary, our results suggest that minks with SBB are ina state of serious oxidative stress. Further studies areneeded to reveal what is the cause of the observed oxidativestress in SSB minks. Moreover, mink may be a suitablemodel to understand the mechanism of SIB in humanbeings or other animals.

Acknowledgments

This study was supported by Doctor Fund provided byNorthwest A&F University. The authors thank Mr. ZhiminZhang and his colleagues at Dalian Jinzhou Mink Farm inLiaoning Province of China for the generous permissionand kind help to collect all of the samples.

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