Subclinical mastitis causes alterations in nitric oxide, total oxidant and antioxidant capacity in cow milk

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Abstract

The aim of this study was to investigate total antioxidant (TAC), and oxidant capacity (TOC) and nitric oxide (NO) levels in milk of cows with subclinical mastitis. Brown Swiss and Holstein breed cows were screened with California Mastitis Test (CMT) to determine mammary glands with subclinical mastitis. Moreover, somatic cell counts (SCC) were determined electronically in all milk samples. Mammary quarters were classified as healthy (n = 25) or subclinical mastitis (n = 35) based on CMT scores and somatic cell count (SCC: ⩽200,000/ml or >200,000/ml) in milk. Nitric oxide, TOC and SCC levels were significantly higher (p < 0.001, p < 0.005 and p < 0.001, respectively) in milk from mammary quarters with subclinical mastitis compared to those from healthy mammary quarters. In conclusion, subclinical mastitis results in higher NO concentrations, TOC and SCC, and NO and TOC were positively correlated with SCC. Moreover, alterations in NO levels and TOC in milk could be used as an alternative diagnostic tool to screen for subclinical mastitis.

Introduction

Mastitis is a common disease that decreases milk yield and quality in lactating cows (Philpot and Nickerson, 1991a, Christ et al., 1983). Because of higher treatment cost and higher culling rate, mastitis causes economical losses (Moore et al., 1991, Philpot and Nickerson, 1991b). In addition to control of clinical mastitis, earlier diagnose and treatment of subclinical mastitis, which is spread unrecognizably in a herd leading to lower milk production, is also required (Craven, 1987, Egan, 1995, Yalcin et al., 2000). Somatic cell count (SCC) in milk is a well known indicator reflecting mammary health and milk quality. Excessive amounts of neutrophiles, macrophages, lymphocytes, eosinophiles and various epithelial cells of mammary tissue in milk is considered as a response of mammary tissue to microorganisms in part of inflammation of mammary gland (Knnapen et al., 1999, Smith, 1994, Weiss, 1989). The level of cytokines such as TNF-α, IL-1β, IL-6, IL-8 and some other molecules such as nitric oxide (NO) are reported to increase during infections (Moilanen and Vapaatalo, 1995, Notebaerta et al., 2008, Riollet et al., 2000). Nitric oxide is produced from l-arginine by nitric oxide synthase (NOS) (Moncada et al., 1991). Two calcium-binded isoforms of NOS are generally present in endothelial cells and brain (Bredt and Snyder, 1994). The inducible isoform of NOS (iNOS) is generally present in macrophages, and production of NO during inflammatory diseases is quite higher (MacMicking et al., 1997, Moilanen and Vapaatalo, 1995). NO, produced in higher amount during inflammation by iNOS and activated by cytokines, is accepted as a primer defense system (Huie and Padmaja, 1993, Okamoto et al., 1997). Antimicrobial effect of NO on bacteria is due to peroxynitrite, a reactive nitrogen metabolite, derived from oxidation of NO (Beckman et al., 1990). Substances, such as peroxynitrite, cause alterations in antioxidant balance in the organism (Chaiyotwittayakun et al., 2002). In the presence of inflammation, proinflammatory cytokines and cytotoxic radicals released from phagocytic cells (Knnapen et al., 1999) result in inhibition of cellular metabolic pathways and lipid peroxidation (Goff et al., 1996). Previous studies revealed that increase in lipid peroxidation during mastitis causes a decrease in levels of some antioxidant molecules leading to an increase in oxidative stress (Goff et al., 1996, Komine et al., 2004, Weiss et al., 2004). Oxidative stress is generally described as an imbalance between oxidant and antioxidant levels (Richter-Landsberg and Vollgraf, 1998, Lykkesfeldt and Svendsen, 2007). Oxidative stress is commonly observed in different pathological events such as pneumonia, sepsis and mastitis of farm animals (Basu and Eriksson, 2001, Lauritzen et al., 2003, Lykkesfeldt and Svendsen, 2007). When the cellular oxidant state is overwhelmed by excessive production of reactive oxygen species and the condition may end up with cellular damage due to oxidative stress and lipid peroxidation (Chaiyotwittayakun et al., 2002, Halliwell and Gutteridge, 1999). Therefore, earlier diagnosis and treatment of diseases, such as mastitis, is important to minimize economical losses. Although it is well known that mastitis increases epithelial cells in milk (Philpot and Nickerson, 1991c), alterations in biochemical composition of milk due to mastitis is not well known. Consequently, the objectives of this study were to investigate nitric oxide (NO) levels, total antioxidant (TAC) and oxidant capacity (TOC) in milk of cows with subclinical mastitis in this study.

Section snippets

Animals and clinical examination

This study was conducted in cows (Brown Swiss and Holstein) under the same nutrition and management conditions at Atatürk University, Veterinary Faculty, Research and Training Farm. All mammary quarters were screened with California Mastitis Test (CMT) to investigate healthy mammary quarters and mammary quarters with subclinical mastitis. Following washing and drying the mammary teats, 70% ethanol was sprayed and a few streams of milk were discarded. Afterwards, milk samples (10 ml) were

Results

For milk samples from mammary glands with and without subclinical mastitis, NO concentrations, TAC, TOC and SCC were 8.89 ± 0.89, 3.96 ± 0.44 μmol/L; 0.42 ± 0.047, 0.54 ± 0.051 mmol TroloxEquv./L; 20.88 ± 0.90, 15.91 ± 0.57 μmol H2O2 Equv/L; (859.46 ± 173.73) × 1000/ml, (87.41 ± 9.53) × 1000/ml, respectively (Table 1). NO and TOC levels were significantly (p < 0.001, p < 0.005, respectively) higher in milk samples from mammary glands with subclinical mastitis compared to mammary glands without subclinical mastitis. In

Discussion

Nitric oxide is a molecule regulating many biological functions in the body and plays some important roles during inflammatory process (Dawson and Dawson, 1995). It has been reported that mammary gland epithelial cells and macrophages produce NO in significant amounts during inflammation (Bouchard et al., 1999, Cuzzoerea and Caputi, 1999, Goff et al., 1996). Mastitis is caused by inadequate hygiene, stress, trauma, improper milking techniques and housing conditions leading to inflammation in a

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    This study was supported by Kafkas University Research Fund (2008-VF-26).

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