The level of oxidative stress and antioxidant status in the sub-clinical ketosis were determined as a mean to understand the increase in susceptibility to sub-clinical mastitis. Of the seventy samples of milk and urine collected from the suspected cases of sub-clinical ketosis, fifty samples were found positive for the presence of ketone bodies which confirms the condition of sub-clinical ketosis. Among the above fifty samples, twenty five samples were found positive for sub-clinical mastitis in terms of somatic cell count and bacterial culture. Lipid peroxides and the activities of superoxide dismutase, catalase, and glutathione peroxidise were significantly more in those twenty five cases. The level of glutathione was significantly high where as the level of ascorbic acid is significantly less. Thus, in the condition of sub-clinical ketosis, the alteration of antioxidant status and existence of oxidative stress can make the peri-partureant animals susceptible to infectious diseases.
Ketosis is a metabolic disease commonly associated with the high producing dairy cows that occurs during transition period. It is caused by a negative energy balance and typically occurs within two months after calving, even when clinical signs do not appear. In India incidence of ketosis varies from 4.22 to 50%, the highest incidence observed during first 30 days of calving (Sharma, 2010). Ketosis can occur as clinical or subclinical, with each having subgroups of primary and secondary. Ketosis can affect milk production. It has been estimated that sub-clinical ketosis reduces milk yield 300 to 450 kg per lactation (Duffield, 2000). The negative energy balance observed during sub-clinical ketosis condition reported to be associated with increased risk of periparturient diseases like mastitis, metritis etc. Leslie et al., 2000 reported that sub-clinical ketosis was an important risk factor for subsequent occurrence of clinical mastitis. During transition period, alteration in physiological factors associated with increased milk production enhances the energy demand and increases the oxygen requirement (Gitto et al., 2002). Increase in oxygen demand augments the production of Reactive Oxygen Species (ROS). Antioxidant defense system limits the production of free radicals and resultant damage at cellular and tissue levels.
Hence, it is planned to study the oxidative stress and antioxidant status during sub-clinical ketosis to understand the mechanism of increased disease susceptibility observed during sub-clinical ketosis.
Materials and Methods
Collection of Samples and Screening for Sub-clinical Ketosis
Venous blood was collected from 15 normal cows (apparently healthy) and 70 crossbred cows with the suspected sub-clinical ketosis within one month of lactation. The guidelines of institutional animal ethical committee were followed while collecting samples from the animals. Cows were all crossbred Jersey and HF dairy cows of three to four years of age. Whole blood (5 ml) with anticoagulant was used for measuring antioxidant status and lipid peroxides. Urine samples were collected and Rothera’s test was performed to detect the presence of acetone and acetoacetic acid. Milk sample were collected from the above animals and tested for the presence of -hydroxy butyrate (BHB) levels by using milk BHB kit (Porta BHB milk ketone test). The concentration of milk BHB greater than 500+ µmol/L was considered to be indicative of sub-clinical ketosis. The milk samples found positive for sub-clinical ketosis were subjected for California Mastitis Test (CMT) and bacterial culture to identify the sub-clinical mastitis.
Oxidative Stress and Antioxidants
The level of oxidative stress was determined based on the level of lipid peroxides (Esterbauer and Cheeseman, 1990) in plasma and the level of antioxidants were also measured as per the methods indicated using hemolysate prepared – superoxide dismutase (Marklund and Marklund, 1974), catalase (Aebi, 1984), glutathione peroxidise (Rotruck et al., 1973), glutathione (Tietze, 1969) and ascorbic acid (plasma) (Jagota and Dani, 1982).
The mean and standard deviation obtained for each estimation were calculated and subjected for statistical analysis using unpaired Student t-test. The significance of estimation was measured at 5% (P<0.05) and 10% (P<0.01) level.
Results and Discussion
Out of seventy samples, each of milk and urine collected from suspected cases of sub-clinical ketosis, fifty samples were found positive for sub-clinical ketosis in terms of presence of acetone/acetoacetic acid in urine and presence of -hydroxy butyrate in milk. On examination of these fifty samples for somatic cell count and bacterial culture, forty three samples were found positive for increased somatic cell count and twenty five samples were positive for bacterial culture. The samples obtained from age matched, apparently healthy controls (15 no.) were found negative for CMT test and bacterial culture.
Negative energy balance observed during transition period is mainly responsible for bovine ketosis. Due to negative energy balance, fat from the adipose tissues are mobilized and increases the level of non-esterified fatty acids. The free fatty acids can disrupt the mitochondrial function by uncoupling the oxidative phosphorylation and thereby helps in the generation of ROS as demonstrated by Bakker et al. (2000). Further, increase in metabolic demand observed during late pregnancy, parturition and initiation of lactation can lead to the increase in the production of ROS (Sordillo, 2005). Free radicals formed during oxidative stress can lead to the peroxidation of poly unsaturated fatty acids (Kumaraguruparan et al., 2002). The severity of oxidative stress can be evaluated by determining the level of lipid peroxides (Halliwell and Whiteman, 2004). In the present study the level of lipid peroxides (Table 1) was found to be significantly (P<0.01) higher in the case of sub-clinical ketosis when compared to that of control animals. Therefore, the existence of oxidative stress condition during sub-clinical ketosis is confirmed.
Table 1: Levels of lipid peroxides and antioxidants
|Name of the Analyte||Control||Subclinical Ketosis|
|(mean ±SE)||(mean ±SE)|
|Lipid peroxides (nmoles/dl)||14.37 ± 4.94||75.63 ± 25.62**|
|Superoxide dismutase (U/ml)||14.89 ± 0.87||18.54 ± 0.95**|
|Catalase (µmoles/min/mg protein)||0.157 ± 0.024||0.909 ± 0.257**|
|Glutathione Peroxidase(μg of GSH consumed/ min/mg protein )||0.059 ±0.058||0.403 ± 0.106**|
|Glutathione (µg/dl)||2549.13 ± 874.01||2885.54 ± 600.16*|
|Ascorbic acid (mg/dl)||4.53 ± 0.41||2.98 ± 0.76**|
Associated with the oxidative stress condition, the levels of activity of superoxide dismutase, catalase and glutathione peroxidase were significantly (P<0.01) higher in the case of sub-clinical ketosis (Table 1). In addition, the level of non-enzymatic antioxidant, glutathione level was significantly (P<0.05) increased in sub-clinical ketosis, whereas the level of ascorbic acid was found to be significantly (P<0.01) low when compared to the control animals (Table 1).
Antioxidant potential of peripaturent cows are found to be altered in relation to physiological changes of parturition (Bernabucci et al., 2005; Sordillo et al., 2007). Sahoo et al., 2009 reported increased activity of superoxide dismutase and catalase during oxidative stress. According to the study reported by Rezapour and Roudbaneh (2011), the level of lipid peroxidation is increased along with the increase in the activity of superoxide dismutase and glutathione peroxidise in RBC with associated increase in -hydroxy butyrate and non-esterified fatty acid levels. Krishna Mohan and Venkataramana (2007) reported that the activity of superoxide dismutase and glutathione peroxidase found to be increased during parturition. The activity of glutathione peroxidase is found to be increased during early lactation (Aitken et al., 2009). As ascorbic acid in animal body is derived from only glucose, high producing cows are known to contain low level of ascorbic acid due to diversion of glucose for the enhanced production of milk (MacLeod et al., 1999). Therefore it is expected to find low level of ascorbic acid as seen in this study.
Thus present study indicate that the cows suffering from sub-clinical ketosis found to be oxidatively stressed along with the altered antioxidant potential which can contribute to the periparturent diseases as it is being supported by the observations of Waller (2000); Gitto et al. (2002) and Sordillo (2005). In addition, the level of -hydroxy butyrate in milk of sub-clinical ketosis condition was found to be significantly higher along with the elevated levels of somatic cell count as indicated by the CMT as well as by the presence of pathogenic micro-organisms as supported by the bacterial culture. Grinberg et al., (2008) have reported that the level of -hydroxy butyrate was higher in milk and found to be associated with increased somatic cell count (SCC) and the presence of pathogenic bacteria. Further, the study has confirmed that -hydroxy butyrate had an adverse effect on neutrophil function. When bovine neutrophils are incubated with the level of -hydroxy butyrate similar to that of an animal in ketosis, the phagocytosis of E.coli is reduced fivefold. Therefore, the initial response of the immune system in the mammary gland to an invading pathogen is severely compromised in the presence of -hydroxy butyrate.
Thus it is concluded that the condition of oxidative stress along with the altered antioxidant status and elevated levels of -hydroxy butyrate in milk observed during sub-clinical ketosis condition favours the disease susceptibility as supported by the existence of sub-clinical mastitis condition which can further affect production of animals.
Conflict of Interest
The authors declare no conflict of interest.
The authors gratefully acknowledge the Dean, RIVER for the facilities to conduct this research work.
Jacob Thanislass conceived and planned the research work and written the article. Pakkiri Muralidaranae conducted the research work.