Antioxidants Profile, Oxidative Stress status, Leukogram and Selected Biochemical indicators in Dairy Cows affected with Mastitis

| This study was designed to evaluate the alterations in oxidative stress status, antioxidants profile, leucogram and some biochemical parameters in Holstein-Friesian (H-F) dairy cows suffered from acute clinical mastitis. Thirty-nine lactating H-F cows were included in this experiment. According to the findings of the clinical examination, the animals were allocated into 2 groups. The first group consisted of 26 cows affected with acute clinical mastitis, while the 2 nd group consisted of 13 apparently healthy cows with negative reactions to California mastitis test (CMT) were served as control group. Physical examination of the selected animals including milk and udder was done. Blood, serum and plasma samples were obtained. Antioxidants, Oxidative stress, leucogram and some biochemical indicators were estimated. Malondialdehyde (MDA) level was significantly increased (P<0.05) in mastitic cows than controls along with nonsignificant increase (P>0.05) in the hydrogen peroxide levels in the same group. Catalase (CAT) activities and total antioxidant capacity (TAC) levels were significantly decreased (P<0.05) in the mastitic group meanwhile, glutathione-s-transferase (GST) activities were insignificantly reduced in the same group. Significant elevated values (P<0.05) of total leucocytic count (TLC) along with significant reductions (P<0.05) in lymphocytes were observed in mastitic cows. Significantly reduced levels (P<0.05) of total protein (TP), globulin, calcium and phosphorus were recorded in the mastitic group as compared to controls. In summary, acute clinical mastitis in H-F cows is obviously associated with marked systemic involvement, typically severely swollen painful udder, and abnormal milk. It is also complemented with significant oxidant/antioxidant, biochemical and leukogram variations that could be used as useful indicators for dairy cows pathological status (mastitis). Oxidant-antioxidant changes explained that cows affected with acute mastitis are accompanied by marked oxidative stress that could be used as a biomarker to check the udder health status.

total antioxidant capacity during clinical and subclinical mastitis in dairy cattle was reported (Atakisi et al., 2010) with eventual occurrence of oxidative stress (Lykkesfeldt and Svendsen, 2007). Oxidative stress commences when the generation of reactive oxygen species (ROS) surpasses the antioxidant defense capacity with resultant oxidative injury to macromolecules including DNA, lipids and proteins (Sordillo and Aitken, 2009). Moreover, ROS could reduce the immune system response toward infection through its harmful effect on the immune cells (Spears and Weiss, 2008). Alterations in the blood oxidative stress markers including reduced glutathione (GSH), lipid peroxides, trace elements and vitamins during mastitis have been reported (Ranjan et al., 2005;Kizil et al., 2007). However, published data about the enzymatic antioxidants in mastitic cows is still scanty ( Jhambh et al., 2013). Enzymatic antioxidants are the most effective direct mechanism in catalyzing ROS (Sordillo and Aitken, 2009). Accurate diagnosis is mostly supported by examining the hematological and biochemical markers of animals. Aberrations from the normal hematobiochemi cal parameters is indicative of disease condition, in addition they aid in the diagnosis and prognosis of illness (Garba et al., 2019). Moreover, it has been realized that leucogram is an essential and reliable standard in assess ing the animal health status (Blumenreich, 1990). So, the current work was designed to assess antioxidants profile, leukogram, oxidative stress status and some selected biochemical parameters alterations in lactating Holstein-Friesian cows affected with acute clinical mastitis.

animals and ExpErimEntal dEsign
The experimental proce dures were approved by the Local Ethics Committee for Animal Experiments. A total of 39 Holstein-Friesian lactating cows, 3-7 years old, in their 2 nd to 5 th parity, 60-120 days in milk belonging to a private farm in Beni-Suef province, Egypt were enrolled in this study. The cows were housed in free stall barns under the same managemental and nutritional conditions. Cows fed a total mixed ration and automatically milked twice daily in a tandem type milk parlor. Selected cows were separated into two groups; the first group (mastitic cows) consisted of 26 cows with acute clinical mastitis, and the 2 nd group (control group) have 13 apparently healthy cows free from external, internal and blood parasites with negative results to California mastitis test (CMT).

CliniCal Examination
All animals were thoroughly examined (Radostits et al., 2000) and the clinical findings were recorded. Udder examination included inspection and palpation of the udder to ascertain the size and consistency of various quarters as well as if any possible abnormalities were present. Manual palpation of the supramammary lymph nodes was done for determining its size and if any indurations were observed.

blood sampling
Under aseptic conditions, two blood samples were collected from each cow through jugular vein puncture. Samples on anticoagulant (EDTA) treated tubes were used for leucogram profile examination and the remained part of the sample was used to separate clear plasma. Plasma samples were stored at -20°C until the estimation of antioxidant profile. The second blood sample was obtained in plain tubes to harvest clear non-hemolyzed serum that was stored at -20°C until estimation of malondialdehyde (MDA) and biochemical parameters.

antioxidant-oxidant biomarkErs
Plasma total antioxidant capacity (TAC) was assessed according to the method defined by Koracevic et al. (2001). Plasma glutathione-s-transferase (GST) was measured as the standard method of Habig et al. (1974). Plasma catalase (CAT) and hydrogen peroxide (H 2 O 2 ) activities were measured according to the standard method of Aebi (1984). The serum lipid peroxidation concentration was estimated according to the intensity of thiobarbituric acid reactive species (Placer et al., 1966). The amount of the MDA was used as an indicator for lipid peroxidation.

bioCHEmiCal analysEs
Serum glucose, cholesterol, triglycerides, protein profile, calcium and phosphorus levels were estimated spectrophotometrically using available commercial test kits according to the procedures of Burtis and Ashwood (1999). The test kits were supplied by Biodiagnostic (Cairo, Egypt).

HEmatologiCal invEstigations
Leucogram profile examination was done within few hours after sample collection. Total leucocytic count (TLC) was performed by using improved new-Bauer chamber method (Feldman et al., 2000). To estimate differential leucocytic counts, moderately thin blood smears were fixed by methyl alcohol and stained by Gimesa. Relative percentage of one hundred leucocytes was identified (Feldman et al., 2000).

statistiCal analysis
Data were analyzed by student t-test using statistical software package SPSS 22. The comparison between mean values was done at 5 % significance level. The results were expressed as mean±SD.

Journal of Animal Health and Production
December 2020 | Volume 8 | Issue 4 | Page 185 milk production as well as udder inflammation findings (Swelling, redness, hotness). The supramammary nodes were enlarged and the affected quarters of the udder were swelled, hot, hyperemic and firm in consistency. The excreted milk from the mastitic cows showed physical abnormalities such as discoloration, flakes and abnormalities in consistency. Meanwhile, neither of the above signs were recorded in the control cows which showed normal excreted milk with negative CMT reaction and absence of udder inflammatory signs.
Concerning the oxidant-antioxidant biomarkers (Table 1), it showed that TAC and catalase levels were significantly decreased (P<0.05) in mastitic group meanwhile, GST activities were non-significantly reduced (P>0.05) in the same group. MDA levels were increased significantly (P<0.05) in mastitic cows as compared to healthy control group, but hydrogen peroxide levels were non-significantly increased (P>0.05) in the in the same group.  Regarding the serum biochemical parameters (Table 2), non-significant (P>0.05) reduced levels of blood serum glucose, cholesterol and triglycerides were recorded in mastitic group meanwhile, total protein, globulin, calcium and phosphorus levels showed significant reductions (P<0.05) in the same group.
Leucogram results were summarized in Table 3. WBCs counts increased significantly (P<0.05) in diseased group as compared to controls. Non-significant (P>0.05) higher neutrophilic and monocytic percentages were observed in mastitic group. Lymphocytes and eosinophils were reduced in diseased group and the reduction was significant concerning lymphocytes (P<0.05).

DIScuSSIon
Mastitis could be defined as inflammation of the parenchyma of the udder tissue regardless the cause (Radostits et al., 2006). It induces physical and chemical alterations in the excreted milk as well as pathological changes in the mammary gland (Babaei et al., 2007). In the current study, the cows of the mastitic group showed elevated body temperature, anorexia, dullness, decreased milk yield as well as inflammatory udder signs including redness, swelling, hotness and pain reaction. Moreover, the excreted milk showed physical abnormalities such as presence of discoloration, flakes, abnormalities in consistency and sometimes contained purulent materials. Nevertheless, neither of these findings were recorded in the control cows. Such clinical findings are consistent with Radostits et al. (2006).
Free radicals are naturally generated due to the intensive metabolism that occurs in the cells of all living organism, especially dairy cows. Oxidative stress commences when a disturbance occurs in the homeostasis which is caused principally by production and accumulation of the free radicals which eventually could predispose the dairy cows to mastitis. Antioxidants conserve the body from oxidative damage of these free radicals either by direct scavenging of them or by prohibiting the oxidizing enzymes activity (Abd-Ellah, 2010). The current study revealed that acute clinical mastitis is accompanied with a compromised antioxidant defense. This could be revealed through the significant increase in MDA levels along with the significant decrease

Journal of Animal Health and Production
December 2020 | Volume 8 | Issue 4 | Page 186 in TAC and CAT levels. Such findings could indirectly indicate elevated free radical activity that reflect the state of oxidative stress that occurs in such cases (Celi, 2011). Catalase activity was significantly decreased (P<0.05) in mastitic group. Similar results were previously reported (Sharma et al., 2010;Jhambh et al., 2013). Decreased antioxidant enzymatic activity could be attributed to the increased consumption to neutralize ROS generated from the inflamed gland, indicating a compromised antioxidant defense mechanism ( Jhambh et al., 2013).
In the present study, GST activities were non-significantly reduced in the mastitic group. Such reduced activities could be attributed to the depletion of the enzyme in neutralizing the excessively produced ROS from the inflamed mammary gland (Zigo et al., 2019).
Estimating the status of lipid peroxidation is one of the most commonly used methods for determining oxidative stress (Kohen and Nyska, 2002). Lipids, mainly phospholipids, that contain polyunsaturated fatty acids are essential components of the cellular membrane's lipid bilayer that are readily oxidized (Catalá, 2010) with eventual generation of lipid peroxides that are decomposed to aldehydes such as malondialdehyde (MDA), which is easily identified by thiobarbituric acid (Kohen and Nyska, 2002). In this study, a significant increased MDA levels were observed in mastitic cows. Similar results were obtained by (Kizil et al., 2007). This could be attributed to the excessive production of ROS as hydroxyl radicals from the activated neutrophils in the inflamed udder causing peroxidative membrane damage ( Jhambh et al., 2013).
Hydrogen peroxide (H 2 O 2 ) is produced from the dissimilation of superoxide radicals and could be eliminated by glutathione peroxidase and catalase activities (Kohen and Nyska, 2002). The results of the present study revealed insignificant increased levels of H 2 O 2 in the cows affected with clinical mastitis along with significant reduction in CAT activities. These findings indicate a compromised antioxidant defense mechanism that could reflect the state of oxidative stress in the affected cows.
Regarding TP results, a significant decreased level was observed in the mastitic group. Similar results were obtained by Krishnappa et al. (2016). This could be attributed to the reduced albumin levels caused by the immune response associated with udder infection (Singh, 2000). In contrast, Ali et al. (2017) recorded significant increase in the TP levels in clinical and subclinical mastitis affected cows. Shifting to albumin, there was non-significant recorded reduced levels in the mastitic cows. Ali et al. (2017) recorded a significant decreased albumin levels in cows affected with clinical mastitis. They explained hypoalbuminaemia on the account of stress condition that occurs during mastitis which enhance the protein catabolism. Moreover, Heinrich et al. (1990) reported that albumin is known to be a negative acute phase protein. Globulin levels were significantly decreased in mastitic group as compared to healthy controls. Similar results were reported by Ali et al. (2017).
In the present study, non-significant reduced levels of blood serum glucose, cholesterol and triglycerides were recorded in mastitic cows when compared to healthy controls. Similar results were recorded by Ali et al. (2017). Blood serum calcium and phosphorus values were significantly decreased in the mastitic group when compared to control group. Similar results were recorded by El-Zubeir et al. (2005). They explained that the reduced levels could be attributed to the mineral losses from blood stream into the mastitic udder. Moreover, Krishnappa et al. (2016) recorded that lowered phosphorus levels in mastitic animals could be caused by its higher excretion in milk, due to the damaged wall of the udder with consequent increased loss in milk.
In the contrary, Das et al. (2018), recorded elevated serum calcium levels in mastitic cows and they explained that to the reduced milk yield in affected animals which causes lowered calcium excretion in milk.
In the current study TLC count increased significantly (P<0.05) in mastitic cows along with non-significant higher neutrophilic and monocytic percentages. Lymphocytic and eosinophilic counts were reduced in the mastitic cows. Elevated total leukocytic and neutrophilic counts along with lymphopenia in mastitic cows was reported by Sarvesha et al. (2017). The significant rise in WBC and neutrophilic counts in mastitic cows may be caused by various immunomodulatory effects (Bagnicka et al., 2011). Moreover, Abba et al. (2013) explained the increased value of neutrophils to the chemotactic factors that are released by the infectious agents in addition to other components of the immune system that trigger the recruitment of neutrophils to the sites of infection.

concLuSIon
Acute clinical mastitis in dairy cows is associated with significant alterations in oxidant/antioxidant status, leukogram and biochemical indicators. A significantly increased TLC and significantly decreased calcium, phosphorus and globulin levels were observed in mastitic cows in this study. These observations could be used as a useful index for mastitis in dairy cows. Furthermore, elevated oxidative stress biomarkers with diminished antioxidant defense that were recorded in current study declared that dairy cows affected with acute mastitis have a pronounced oxidative stress. This necessitates using of antioxidant therapy along with the conventional mastitis treatments in ameliorating the oxidative damage that occurs in the secretory cells and so reducing the subsequent