Impact of The Ageing on Oxidant/Antioxidant Balance And Hematology of Indigenous Cow and Its Amelioration by Ascorbic Acid

Kavitha Krishnappa (  geturkavitha@gmail.com ) KVAFSU Bangalore: Karnataka Veterinary Animal and Fisheries Sciences University Regional Campus Bangalore UMESH DIMRI Indian Veterinary Research Institute MAHENDRAN KARUNANITHY Indian Veterinary Research Institute SHANKER K. SINGH UP College of Veterinary Science and Animal Husbandry Mathura ALOK KUMAR CHAUDHARY UP College of Veterinary Science and Animal Husbandry Mathura GYANENDRA KUMAR GAUR Indian Veterinary Research Institute GYANENDRA SINGH Indian Veterinary Research Institute MEGHA PANDE IVRI: Indian Veterinary Research Institute Vivek Joshi Indian Veterinary Research Institute Med Ram Verma Indian Veterinary Research Institute Harini Kallambella Ramakrishne Gowda Indian Veterinary Research Institute Stanzin Angmo Indian Veterinary Research Institute


Introduction
The livestock sector plays a very important role in the welfare of Indian rural economy. As age progresses, most vital organs show a gradual deterioration in their functional capacity attributed to the marked reduction in the individual's reserve capacity. To understand the ageing process, several hypotheses have been postulated. But, the evidences suggest that the harmful and cumulative effects of the reactive oxygen species (ROS) during lifespan are the paramount factors of the ageing process (Knight, 2000;Junqueira et al., 2004). An elevated release of free radicals by aging mitochondria and reduced antioxidant defense of geriatric individuals probably attributed to the development oxidative stress (Harman, 1998;Finkel and Holbrook, 2000). Oxidative changes in lipids, proteins and DNA have been produced by excessive levels of ROS and other reactive oxidants in aged animals (Chen et al, 2000).
However, the studies demonstrating the associations of oxidative stress with ageing are still not clear (Cini and Moretti, 1995). The role of free radical reactions is now widely recognized in the clinical pathology of many diseases of cattle; however, the potential role of these reactions in the ageing process of indigenous cows is not yet unraveled.
Hematology always be helpful to assess the animal's health status and values of haematological panels alter as the age advances. A decreased haemoglobin, PCV and leukocyte counts is observed with the advancement of the age of animals. Routine haematological appraisal of geriatric animals could assist in earlier detection of degenerative diseases (Dell'Osa and Jaensch, 2016).
Oxidative stress is characterized by the disproportion between the development of oxidants or free radicals and the capability of the body to use anti-oxidant protection mechanisms to eliminate its adverse effects. Oxidative parameters SOD, CAT and GSH, along with GPX, are the most essential intracellular antioxidant defense mechanisms to counteract the imminent oxidative damage caused by free radicals during in ammation or other triggering agents. As postulated by Harman (1998) in his free radical theory of ageing, lipid peroxidation plays a crucial role in ageing individuals. Increased oxidative stress is sequel to production and exposure to ROS and antioxidant defense mechanism by the host in turn will lead to senescence (Stadtman, 2004).
In both animal and human medicine, the role antioxidants in health and various disease conditions is demonstrated very well (Valko et al., 2007;Celi, 2011). Brie y, antioxidants (such as thiols, ascorbates and polyphenols) can act against ROS and hence prevent oxidation. In most mammalian species except human, non-human primates and guinea pigs, ascorbic acid, a six-carbon lactone is synthesized from glucose in the liver. In blood plasma, neutrophils and other body tissues, ascorbic acid is an important antioxidant nutrient and has numerous physiological functions by playing a signi cant role in controlling the immune functions and providing protection at several cellular and subcellular stages from oxidative damage. Levels of ascorbic acid usually decreased during stress and physiological dysfunction as reported by several researchers (Ali, 2000;Ranjan, et al. 2005).
Most of the studies focused on the use of immunomodulators and antioxidants in therapeutic and prophylactic management of infectious diseases. However, there is a scarcity of information on oxidant/antioxidant pro le and use of antioxidants in apparently healthy geriatric animals. Moreover, gerontological studies in livestock farm animals are meager as compared to pet and laboratory animals.
Keeping in view of these facts and research reports, the current study was undertaken to study agerelated alterations in oxidant/antioxidant balance and hematological panels of apparently healthy Tharparkar cattle. The evaluation of bene cial effects of ascorbic acid supplementation against altered oxidant/antioxidant balance and hematological panels in ageing was also aimed.

Experimental design
The research was conducted at the LPM section of (cattle and buffalo farm) Indian Veterinary Research Institute, ICAR-IVRI, Izatnagar, Uttar Pradesh 243 122 India. Selection of animals was made randomly from the farm herd, Tharparkar breed was selected for the study which is maintained under common nutrition and managemental strategies. Animals were fed on balanced ration prepared at the Feed Technology Division of the Institute. There was no additional feeding of antioxidant such as vitamin E and selenium. The animals were categorized into 4 groups (groups 1 to 4), each group having 6 animals (n=6). Groups were based on age in years. Group I consisted of birth to less than one year, II consisted of As age-related variations in the indices of oxidative stress and hematological parameters were observed, ameliorative steps were undertaken using ascorbic acid. All the groups were treated with commercial preparations of ascorbic acid @ 20mg/kg-SID i/m for 5 consecutive days. The dose of ascorbic acid was determined according to Roth and Kaeberle (1985) report.

Sample collection and processing
Animals were evaluated for changes in oxidant-antioxidant pro le and hematological parameters before the start of therapy (day 0) and post therapy (day 6, 12, 18 and 24) of the study. Blood samples (10 ml) were collected from the jugular vein puncture in vials containing heparin and EDTA. The heparinized blood was centrifuged at 3000 rpm for 10 min and plasma and buffy coat were removed. In Phosphate Buffered Saline (PBS, p H 7.4) solution, the RBC's were gently dissolved and centrifuged again at 3000 rpm for 10 min. This process was repeated twice after discarding supernatant. About 2/3rd of the RBC pellet was diluted with ice-cold distilled water in 1:10 ratio for the preparation of 10% hemolysate, which was used for the estimation of lipid peroxidation (LPO), superoxide dismutase (SOD) catalase (CAT) and glutathione peroxidase (GPX). Remaining 1/3rd of the RBC pellet was diluted with PBS solution in 1:1 ratio to get RBC suspension which was used for reduced glutathione (GSH) estimation. Cyanohemoglobin procedure was used to measure haemoglobin concentration of hemolysate (Tentori and Salvati, 1981).

Evaluation of oxidant-antioxidant pro le
According to Placer et al., (1966) lipid peroxidation concentration in RBC hemolysate in terms of concentration of malondialdehyde (MDA) per mg haemoglobin was measured. As per Prins and Loos, (1969) reduced glutathione in RBC suspension was measured by DTNB (5, 5 ' -Dihiobis-2-Nitro Benzoic acid) method. The levels of superoxide dismutase (SOD) were determined according to the Madesh and Balasubramanian (1998) method and the unit is expressed as µmol MTT formazan formed per mg of haemoglobin. As per Aebi, (1986) catalase levels in haemolysate were estimated using H 2 O 2 as a substrate. Glutathione peroxidase (GPX) was measured by Paglia and Valentine (1967) method.

Evaluation of hematological pro le
Haemoglobin concentration (gm/dl) in the whole blood was estimated by modi ed Sahlis acid hematin method (Berman, 1919). Jain (1986) method was used for TEC, TLC, DLC (Differential Leukocyte Count), PCV and Platelet count examination.

Statistical analysis
Data were analyzed by two way repeated measures ANOVA. The values were expressed as mean ± S.E. To study the effect of groups, periods and interaction of group & period for various parameters for the repeated observations on the same animals during different periods following general linear model was used. y ijk = μ + G i + ID(G i ) + P j + (GP) ij + e ijk Where, y ijk = k-th observed value of the response variable for i-th group for the j-th period μ = General mean effect ID(G i ) = Random effect G i = Effect of i-th group P j = Effect of the j-th period (GP) ij = Interaction effect of i-th group and j-th period e ijk = error term The multiple comparisons between groups, periods, time intervals and their interactions for various parameters were done by using Tukey test at 5% level of signi cance. The analysis was done by JMP 9.0 software.

Results
Marked oxidative stress was observed in groups 1 to 8 year, 8 to 10 year and above 10 year age group of cattle. Variation in oxidative stress indices with ageing in cattle during the study period is depicted in the table 1. LPO and SOD levels were found to be markedly increased with ageing, and it was signi cantly (P < 0.05) altered in all studied groups of the cows. GSH level was estimated to be decreased with ageing; however signi cant difference between 8-10 years cows and cows above 10 years age was not estimated. Similarly GPX level also found to be decreased with ageing, and signi cant difference (P≤0.05) in GPX level was estimated in 1 to 8 years, 8 to 10 years, and above 10 years age group cows as compared with the animals below one year. The catalase activity of 8 to 10 years and above 10 years cows was signi cantly (P≤0.05) higher as compared to animals below one year age group.
Moreover, mild alterations in the hematological parameters of different age groups of cows were recorded. Values of haemoglobin, TEC, PCV, TLC, differential leukocyte and platelet count are depicted in Table 2 and 3. Below one year aged animals revealed signi cantly (P < 0.05) higher levels of hemoglobin, PCV and TEC concentration compared to other age groups. Signi cant (P < 0.05) reduction in hemoglobin levels with ageing was estimated between 8 to 10 years and above 10 years age in comparison to below one year aged animals. Similarly PCV, TEC and TLC levels were found to be signi cantly (P < 0.05) lowered with ageing. Moreover, 8 to 10 years and above 10 years age group cows revealed signi cantly (P < 0.05) lowered PCV, TEC and TLC values as compared with animals below one year age group.Even though values were within the normal reference range decreasing trend was observed in haemoglobin, TEC, PCV and TLC counts with ageing.
Signi cant (P < 0.05) increase in platelets count was found only in the cows above 10 years age group. In differential leukocyte count signi cant (P < 0.05) reduction in lymphocyte percentage was noticed in 8 to 10 years and above 10 year age group cows, and signi cant (P < 0.05) increase in neutrophils percentage was observed in 8 to 10 year and above 10 year age group animals. Whereas eosinophil percentage was signi cantly higher in above 10 year age group cows. Monocytes and basophil percentage were not found to be signi cantly altered among the various age groups.
After ascorbic acid administration, below one year aged animals did not exhibit any signi cant changes in oxidant/ antioxidant balance and hematological values on days 6, 12, 18 and 24 post-administration. In 1 to 8 year age group animals, signi cant (P < 0.05) reduction in LPO levels was noticed on day 18 and day 24, and the values were comparable to below one year age group. The 8 to 10 years and above 10 years age group cows also exhibited signi cant (P < 0.05) reduction in LPO levels on day 12 and at subsequent interval of collection compared to their day 0 values. In SOD activities, signi cant (P < 0.05) reduction was noticed on day 6 in 1 to 8 years age group, on day 12 in 8 to 10 years and above 10 years age group.
The GSH levels of 1 to 8 years, 8 to 10 years and above 10 years age group cows signi cant (P < 0.05) improvements on days 24, 12 and 6, respectively, was found when compared with their day 0 values. In the both 8 to 10 years and above 10 years age groups, GSH levels were comparable to 1 to 8 years age group cows on day 24. Signi cant (P < 0.05) increment in GPX activities was recorded on day 6 in 1 to 8 years, 8 to 10 years and above 10 years age groups cattle. 1 to 8 year aged cattle revealed a comparable GPX activity as of below one year age group on days 18 and 24. Signi cant (P < 0.05) reduction in catalase activity was observed on day 6 only in above 10 years age group cows.
After ascorbic acid administration, signi cant (P < 0.05) improvement in hemoglobin concentration was observed in 8 to 10 years and above 10 years age group of cattle on day 24. However changes in PCV and TEC levels were non-signi cant, but improved values were observed in 1-8 years, 8 to 10 years and above 10 years age group of cows on day 24 with increasing trend. Signi cant (P < 0.05) elevation in TLC counts was observed on day 12 in 8 to 10 years age and above 10 years age group of cows. The platelets counts were not signi cantly altered in different age groups even after ascorbic acid administration. Signi cant (P < 0.05) increase in lymphocyte percentage was noticed in 8 to 10 years aged cows on day 18. Whereas, signi cant (P≤0.05) reduction in neutrophil percentage in 8 to 10 years age cows was observed on day 24.
Analysis by repeated measure ANOVA method revealed signi cant (P < 0.05) differences between different age groups, before and after ascorbic administration with respect to the values/activities of LPO, SOD, GSH and GPX. No signi cant alteration in catalase activity was revealed in below one year, 1 to 8 years, 8 to 10 years animals. Further analysis of data revealed signi cant (P < 0.05) changes in hematological parameters such as hemoglobin concentration, PCV, TEC, TLC and DLC before and after treatment. Changes in the parameters are predominant on 18th and on day 24 than on 6th and 12th day of treatment. Geriatric group of cattle (> 10 year) exhibited considerable alteration in oxidant/antioxidant and hematological parameters compared to below one year aged animals.

Discussion
Oxidative stress is associated with ageing and various diseases, including neoplasia, neurological disorders, diabetes and skin disorder (Valko et al., 2007;Singh and Dimri, 2010). Cell components, especially mitochondria and DNA sequences are damaged mainly by accumulation of reactive oxygen species (ROS) that are released by oxidative metabolism over a time (Wang and Wang, 2012;Carri et al., 2015,). High productive cows are usually prone to greater risk of health related issues mainly due to inability to combat ROS generated in highly active metabolic cells (Sordillo, 2009). In the present study, our ndings support the hypothesis that the levels of oxidative stress increases, and hematological parameters get altered during the ageing process. Similar pattern of changes were observed in senescent animal during aging in oxidative stress indices (Cand and Verdetti, 1989;Navarro, et al., 2004).
In the current study, LPO and SOD levels were signi cantly (P < 0.05) increased with ageing in all age groups. Free radicals production leads to oxidative damage of proteins, lipids and nucleotides which in turn may cause dysfunction of nervous system and ultimately neuronal death (Head, 2013). Gala et al. (1996) also reported higher levels of MDA and SOD in aged dogs than young group, greater exposure of old tissues and cells to free radicals and their greater peroxidation rate when cell homogenates are exposed to sudden oxidation also indicate that enzymatic activities in red blood cells are highly age dependent (Kaspoglu, et al., 2001) The GSH level and GPX activities were found to be decreased with ageing. Similarly low GSH levels were observed in aged dogs when compared to young dogs as reported by Gala et al. (1996). Enzyme activity is decreased in old tissues and changed with age, which may be one of the potential causes for decreased enzymatic activities (Remacle, et al., 1992). Another reason may be the in vivo oxygen radical production is depleted in aged animals, the reduction in antioxidant defenses might be considered as a decreased physiological compensatory mechanism rather than adverse change leading to additive oxidative damage (Kaspoglu, et al., 2001).
In general, antioxidant vitamins improve various components of cellular and noncellular immune response. Effective antioxidants include naturally occurring free radical scavenging antioxidant enzymes and also products that potentially enhance the enzymatic activity of GPX, GSH, SOD and CAT enzymes. In adult ruminants ascorbic acid is not an important dietary nutrient; however, advantages have been seen by supplementing ascorbic acid for stressed animals in adverse weather conditions and in health related disorders (Kolb, 1984). Ascorbic acid administration is cost effective and intervene harmful effects of free radicals and also very effective against age related neurological disorders in human (Harrison, 2012).
In the present study, administration of vitamin C had signi cantly (P < 0.05) decreased altered LPO and SOD levels at different intervals. Signi cant (P < 0.05) improvement in GPx and GSH levels was noticed in 1 to 8 year, 8 to 10 year and above 10 year age group cattle. Ascorbic acid is a powerful antioxidant and reducing agent soluble in water that prevents protein, lipid and DNA oxidation (Padayatty et al., 2003). Block, et al. (2003) reported a direct correlation between increased lipid oxidation products and decrease of vitamin C & E levels with ageing. Paolisso, et al. (1998) and Mecocci, et al. (2000) also reported decrease vitamin C levels in plasma is associated with increasing age.
The hematological pro le plays an important diagnostic tool as a bare minimum database module. This may be a good tool for monitoring response to therapy, or as a starting point for preparing a list of differential diagnoses and for nding the severity of the infection. However, data integration is important for the highest diagnostic output (Barger, 2003). The variations in biochemical and haematological parameters helps in the evaluation of the animal's physiological or pathological state (Ahmad et al., 2003).
In our study hematological parameters namely hemoglobin, TEC, Hct % and TLC level decreased with ageing, similar ndings has been reported by Deepak kumar (2019), Lane and Campbell (1969). With ageing there may be reduced red blood cell production and more irreparable damage of red blood cell in cow has been reported. E ciency and enzymatic activity of red cells reduces with ageing which may be the reason for reduced activity. Size of the RBC and the associated Hb composition (MCH) of red blood cell increases where as haematocrit and TEC decreases in aged animals (Shaffer et al., 1981).
In the present study, TLC and number of lymphocytes decreased where as neutrophil and eosinophilic counts increased with ageing, mild neutrophilia are predominant in 8 to 10 year and above 10 year age group cattle, which is similar to ndings of Bedenicki et al. (2014). Reduced lymphocyte count may be one of the causes of immunosenescence. Reduced lymphocyte counts can be directly proportional to CD4 + T cells decline with ageing. Stress leukogram leads to reduction in lymphocyte counts (Radakovich et al., 2017).
After administration of ascorbic acid 8 to 10 year and above 10 year age group cattle exhibited signi cant (P < 0.05) improvement in Hb, TEC and PCV levels. Vitamin C increases the haematocrit through enhanced iron absorption and as such helps in reversal of anemia (Atanasova et al., 2004). Signi cant (P < 0.05) improvement in TLC after therapy in 8 to 10 year and 10 year age group animals were observed on day 12.Our nding is in agreement with Fraser et al. (1980) and Field et al. (2002) who reported animals administered with ascorbic acid have exhibited increased levels of total WBC count in blood circulation. White blood cell activity is enhanced by both vitamin C and E administration in healthy individuals (Jeng et al., 1996). This is at variance with reports, Owu et al. (2016) who reported vitamin C did not signi cantly alter the WBC count in the treated groups.
In DLC, neutrophil levels signi cantly (P < 0.05) decreased in 8 to 10 year on day 24 where as lymphocyte levels signi cantly (P < 0.05) enhanced in 8 to 10 year on day 18.These nding were similar to Babe, (2011) who reported vitamin C has the capability to decrease the percentage of neutrophils and increase the lymphocyte percentage signi cantly in heat stressed sheep. An important function of vitamin C in immune system is to protect the body against foreign invaders. Vitamin C completes its important activity by increasing synthesis of WBC's especially neutrophils which helps to ght against microorganisms such as bacteria and virus. Ascorbic acid is very crucial for the immune system and enhances synthesis of prostaglandin, T-lymphocytes and other WBC's (Eberhard et al., 1989).
Even though platelet count increased with age, signi cant (P < 0.05) enhancement was noticed in above 10 year age group cattle. High count can be due to more synthesis of erythropoietin due to greater demands for oxygen and CO 2 transport mainly because of enhanced metabolic activity or faulty exchange of gases caused by respiratory membrane damage (Zaki et al., 2008).
Altered oxidant/antioxidant status and hematological pro le signi es that 8 to 10 year and above 10 year age are in a state of signi cant oxidative stress. Ageing causes change in the cattle oxidant/ antioxidant balance leading to oxidative stress which is observed in the present study.
In conclusions, ageing is associated with a remarkable alteration in oxidative stress indices and hematological parameters. Supplementation of ascorbic acid enhanced the oxidant-antioxidant status and improved altered hematological parameters in different age groups. Hence, supplementation of ascorbic acid may be helpful in managing cattle of different age group for better response. Furthermore, large scale studies are needed in order to validate the potential use of antioxidant therapy with ageing.

Declarations
This is to declare that the article is original, has not been submitted or published elsewhere, and has the approval of all authors.