Selenium-Yeast Administration Ameliorates the Alterations in Haematological and Oxidative Stress Parameters of Wistar Rats Subjected to Restraint Stress

Okwute M Ochayi¹, Ruth Y. Manjak², Emmanual E Ochai³, Oluwatosin I Oyeniran⁴ and Senol Dane^4*

^*Correspondence: Senol Dane, Department of Physiology, Faculty of Basic Medical Sciences, College of Health Sciences, Nile University of Nigeria, Abuja, Nigeria, Email:

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Introduction

Stress can be term as a classical experience of “wear and tear” on the physiological system emanating from the central nervous system to other systems of the body; where it results in mental and physical health problems [1]. Over the years, hormones such as cortisol and adrenaline are being tagged as classical stress hormones. These hormones have been implicated in the elicitation of oxidative stress and the creation of free radicals [2]. Hormones are chemical messengers transported through the blood to target organs; hence, the first tissue to feel the impact of stress is the blood [3]. The study of this effect is well encapsulated under the study of blood in health and disease via haematological indices; which have been implicated in the deleterious effects induced by stress.

Stress leads to a decrease in red blood cell count (RBC), leucocytes or white blood cell count (WBC), packed cell volume (PCV), and haemoglobin concentration (Hb) [4]. It also affects platelet volume index (MPV), platelet distribution width (PDW), red blood cell distribution width (RDW), and mean corpuscular haemoglobin concentration (MCHC) [5,6]. Considering the implicating roles of stress hormones in the generation of free radicals and oxidative stress which ripple effect might be the alteration of haematological indices observed in the aforementioned studies [2]. There is a need to consider some natural antioxidants or supplements such as selenium-yeast which is a major constituent of most grains and feeds [7].

Some studies have reported the pivotal role of selenium-yeast as a potent antioxidant. Selenium yeast was observed to improve the antioxidant capacity of aquatic animals such as juvenile Eriocheir Sinensis when exposed to stress [8]. It plays a critical role in elevating the antioxidant status of Tibetan sheep as seen in malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) concentrations [9]. Considering the above roles selenium plays in the improvement of antioxidant capacity both aquatic and terrestrial lives, one has to possibly reason its application on haematological parameters during stress. This study aimed towards investigating the effects of exogenous selenium-yeast on some haematological indices in Wistar rats exposed to stress.

Materials and Methods

Animals

Fifteen Wistar rats weighing between (100-150 g) were employed in this experiment, they were gotten from the Department of Human Physiology, Ahmadu Bello University, Zaria, Nigeria, and housed at the same venue for the experiment where they were given rats’ food and distilled water ad libitum. All handling was under the best standard practiced following the rules of Ahmadu Bello University Animal Handling Committee.

Experimental groups

The Wistar rats used; were randomly separated into three groups, consisting of 5 rats each (n=5) namely; Group I, acted as normal control (NCG); and they remained undisturbed during the study. Group II, Selenium-yeast group (SYG); was pre-treated with selenium-yeast at 0.1mg/ kg before restraint stress. Group III, Stressed group (SG); was induced with restraint stress and administered distilled water at 1ml/kg.

Methods

Group II and Group III animals were subjugated to restraint stress for 6 h between the periods of (9:00 h- 15:00 h) for 21 days. Group III rats were administered with 1 ml/kg of distilled water before subjecting them to restraint stress, while Group II rats were pretreated with 0.1 mg/kg of selenium-yeast before restraint. Restraint stress protocol was according to the principle of Kumar et al., 2007 using a wire mesh of dimensions 8 cm (length) X 4 cm (Breadth) X 4 cm (High) for the experiment, and a key lock and fastener were employed to fix the rats in a restrainer from escaping. After the experiment, the rats were anaesthetized and the blood sample was obtained to assess for haematological indices and markers of oxidative stress.

Haematological indices

The blood samples collected were stored in a heparinized tube after rats been anaesthetized under light anaesthesia. An automated blood analyzer was used; this was programmed to count red blood cells or reticulocytes (RBCs), white blood cells or leukocytes (WBCs), and Packed Cell Volume (PCV) (Abacus Junior Vet 5, Austria).

Oxidative stress markers

Lipid peroxidation level was evaluated via the concentration of the level of thiobarbituric-acid (TBA) reactive substance and malondialdehyde (MDA), using the NWLSSTM MDA assay kit (Northwest Life Sciences Specialties, Product NWK-MDA01, Vancouver WA, specificity: Malondialdehyde, Sensitivity: 0.08 μM). This follows the interaction of MDA with TBA; leading to the formation of an MDA-TBA2 compound that absorbs greatly at a wavelength of 532 nm [10].

Superoxide dismutase (SOD) activity

Superoxide dismutase (SOD) activity in the serum of the Wistar rats was assessed using the NWLSS SOD assay kit (product NWK-SOD02, Specificity: Cu/Zn, Mn, and Fe Superoxide Dismutase, Sensitivity: 5 U/mL). This follows the principle of the suppression of autoxidation of hematoxylin as explained in the study of Martins et al [11].

Catalase (CAT) activity

Catalase activity was evaluated utilizing the NWLSS CAT activity assay kit (Product NWKCAT01, Specificity: Catalase, Sensitivity: 6.0 Catalase/mL). The enzyme activity was evaluated following the standard that catalase consumes peroxidases substrate at a wavelength of 240 nm.

Glutathione peroxidase (GPx) activity

The serum concentration of glutathione peroxidase was evaluated employing NWLSSTMc GPx (GPx1) ELISA assay kit (Enzyme-Linked Immunosorbent Assay) (Product NWKGPX02, specificity: Glutathione peroxidase, Sensitivity: 12.5 pg/ml). In this sample, the GPx concentration is evaluated by juxtaposing the 450 nm absorbance of sample wells to the absorbance of a recognized standard [12].

Statistical analysis

The statistical package used was Graphpad Prism version 5.0. (San Diego California, USA).

Data gotten from the study were presented as Mean ± standard error of the mean and analyzed employing the analysis of variance (ANOVA) with Turkey’s posthoc test. When P-value is < 0.05, it was deemed significant.

Results

Red blood cell (RBC) counts

The result as shown in Figure 1 reveals that there is a significant increase in RBC count in Group I (Normal control) having a value of 4.2 cells/μL when compared to Group III (Stress control group) having a value of 3.7 cells/μL and Group II (selenium-yeast group). However, there was an increase in the RBC counts in Group II (selenium-yeast group) when compared with Group III (stress group), although the increase in statically negligible.

Figure 1: Effect of restraint stress on RBC.

Data are presented as Mean ± SEM. a, b=Means with different superscript letters are significantly different (P<0.05). Group, I=Normal control, Group II=selenium-yeast stress group, Group III=restraint stress group.

White blood cell (WBC) or Leucocyte counts

The WBC counts significantly increase in Group I (normal control) (42.2 cells/L) when compared with Group III (stress control) (39 CELLS/L) and Group II (selenium-yeast group) (37 cells/L). In the stress groups one can say selenium-yeast decreases the deleterious effects of stress on WBC with an increase value of WBC count in Group II when compared with Group III, although not statically significant (Figure 2).

Figure 2: Effect of restraint stress in WBC.

Data are presented as Mean ± SEM. a, b, c=Means with different superscript letters are significantly different (P<0.05). Group, I=Normal control, Group II=selenium-yeast stress group, Group III=restraint stress group.

Packed cell volume (PCV)

The result for PCV shows a statistical increase in Group I (Normal control group) 42% when compared with Group III (Stressed group) 35%. However, there was an increase in the PCV of Group II (Selenium-yeast group) 39% when compared with Group III. This might be a possible case of improvement as a result of the prior administration of selenium-yeast (Figure 3).

Figure 3: Effect of restraint stress on PCV.

Data are presented as Mean ± SEM. a, b, c= Means with different superscript letters are significantly different (P < 0.05); Group I=normal control, Group II=selenium-yeast stress group, Group III=restraint stress group.

The selenium-yeast has influence on biomarkers of oxidative stress. The mean values increase in Group II for SOD (2.08 ± 0.08); CAT (44 ± 0.90); GSH (33 ± 1.2); MDA (2.6 ± 0.09) when compared with Group III SOD (1.87 ± 0.08); CAT ( 34 ± 0.62); GSH (25 ± 0.3); MDA ( 2.2 ± 0.07). While in Group I the values for MDA (0.1 ± 0.12) significantly decreases when compared with Group II (2.6 ± 0.09) and Group III (2.2 ± 0.07). The result for MDA indicates level of lipid peroxidation (Table 1).

Table 1: Effects of restraint stress and selenium yeast on biomarkers of oxidative stress.

Data are presented as Mean ± SEM. a, b=means with different superscript letters within rows are significantly different (P<0.05); Group I=normal control, Group II=selenium-yeast stress group, Group III=restraint stress group.

Discussion

Haematological parameters are key indicators of the physiological status of both humans and animals. The effects of stress on red blood cell (RBC) as shown in Figure 1 indicated that stress as a deleterious effects on RBC. The reduction in RBC in the stress group agrees with the findings of a study [13] which stated that stress damages the RBC membrane leading to a reduction in RBC counts. However, this finding disagrees with the report of a recent study [14] which showed that the RBC counts increases during stress; this may be due to the kind of stressor (chemical stressor) applied in their study [14], unlike ours which was a physical stressor. However, the administration of selenium-yeast antioxidant mitigate the decrease observe in the RBC counts induced by stress; this agrees with a study [15], which showed that selenium-yeast at 0.03 g/kg/ BW improves RBC counts in higher animals.

Restraint stress decreases leucocytes counts in Wistar rats. This finding conforms to the report of a study [16], which indicated that oxidative stress alters white blood cell (WBC) structures. Stress is known to reduce WBC count, and this might be due to the sensitivity of B-lymphocytes to glucocorticoid cells and its apoptotic nature [17]. Although findings from this present study shows an increase in the WBC count in the selenium-yeast administered group when compared with the stress group. However, this does not agree with the result of a study [18] which showed that selenium-yeast pretreatment does not affect the total WBC count. Nevertheless, this finding is similar to the result of a study [19] which revealed that seleniumyeast pre-treatment improves WBC counts.

The packed cell volume (PCV) decreases in Group III was the least among the three groups which agreed with the finding from a previous research [20], which showed that stress decreases PCV. However, in the selenium-yeast pre-treated group, there was an improvement in the PCV. This shows that selenium-yeast may decrease the deleterious effects of stress of restraint stress on the PCV.

Considering the haematological changes with response to restraint stress administration, there is a need for further verification on the mechanism by which restraint stress induces these changes, though it has been speculated to be due to its ability to induce oxidative stress. The following biomarkers of oxidative stress were evaluated; MDA, SOD, GPx, and CAT. There exist a significant reduction in SOD, CAT, and GSH in the stress group (SG) relative to the selenium-yeast (SYG) and normal control (NCG). However, the activity of MDA which indicates the level of lipid peroxidation was more in the stressed group compared to the normal control group. These findings correspond to the following studies [21- 23].

Conclusion

Our findings from this study have proven that restraint stress induces oxidative stress, thereby altering RBC, WBC counts, and PCV. These alterations in haematological indices can be ameliorated through pre-treatment with selenium-yeast antioxidants.

Funding

This research was fully funded by authors; we didn’t receive any aid from any other source be it government or nonprofit agencies.

Conflict of Interest

The authors have no conflicts of interest to declare.

Ethical Approval

The ethical approval for this study was obtained from the Animal Ethical Committee of Ahmadu Bello University, Zaria, Nigeria. This study was conducted following the best standard practices of the Animal Ethical Committee.

Author's Contributions

OMO formulated the study. RYM, EEO and OIO conducted the research and provided research materials. OMO, RYM and EEO collected organized, analyzed, and interpreted data. All authors were fully engaged in the actualization of this manuscript.

Acknowledgement

We appreciate the efforts of all laboratory technicians who provided technical support during this study.

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