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Research Article

Biochemical Markers of Ketosis in Dairy Cows at Post-paturient Period: Oxidative Stress Biomarkers and Lipid Profile

W.M. El-Deeb and S.M. El-Bahr
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Background: Oxidative stress biomarkers and lipid profiles were used successfully as prognostic and diagnostic biomarkers of many animal diseases. However, their use in the diagnosis of ketosis in dairy cows at post-paturient period is not completely elucidated. Materials and Methods: Therefore, 25 cows suffered from ketosis at post-paturient period were used in the current study together with 20 healthy cows who served as a control. Blood samples were collected from diseased and healthy animals and the harvested serum were used for determination of oxidative stress biomarkers and the profiles of lipids, protein and enzymes. Results: The obtained results declared that, there was a significant (p≤0.05) increase in the levels of aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), non-esterified free fatty acids (NEFA), β-hydroxylbutyric acids (BHBA), malonaldehyde (MDA) and nitric oxide (NO) in dairy cows affected with ketosis compared to control. Conversely, a significant (p≤0.05) decrease in the levels of glucose, total cholesterol, cholesterol ester, free cholesterol, triacylglycerol (TAG), superoxide dismutase (SOD) and reduced glutathione (GSH) were detected in diseased cows compared to control. Serum BHBA, NEFA, MDA and NO levels were positively correlated with each other’s and inversely correlated with activity of SOD and GSH concentration in cows affected with ketosis. Conclusion: Oxidative stress biomarkers and lipid profiles could be used as promising biomarkers for ketosis in dairy cows at post-paturient period. The antioxidant therapy may useful in the treatment of ketosis in cows at post-paturient period.

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W.M. El-Deeb and S.M. El-Bahr, 2017. Biochemical Markers of Ketosis in Dairy Cows at Post-paturient Period: Oxidative Stress Biomarkers and Lipid Profile. American Journal of Biochemistry and Molecular Biology, 7: 86-90.

DOI: 10.3923/ajbmb.2017.86.90

Received: September 23, 2016; Accepted: January 24, 2017; Published: March 15, 2017


The post-paturient (transition) period is the most critical period in dairy cows1 due to severe economic losses for dairy farmers as results of drop in milk production and high culling rates2,3. This period constitutes 3 weeks before and after parturition4. In this period cows are affected by different metabolic and infectious disease for examples ketosis and mastitis, respectively5. Ketosis is mostly occurred after calving because the stimulus for milk production is at its maximum and the demand of the mammary gland for glucose is often greater than the glucose available in blood creating negative energy balance. Negative energy balance stimulate the secretion of hormone sensitive lipase, triggering lipolysis with subsequent release of non-esterified fatty acids from adipose tissues (NEFA) into blood in a bioprocess named lipid mobilization6. Incomplete oxidation of NEFA in the liver resulted in ketone bodies formation (acetoacetate, β-hydroxybutyrate and acetone) and subsequent ketosis7. The most common ketone body in dairy cows is β-hydroxybutyric acid (BHBA). Therefore, NEFA and BHBA are the most common biomarkers for evaluation of ketosis and lipid mobilization8. Lipid peroxidation also may be induced as a result of intensified oxidation of NEFA in liver9. The MDA levels in blood and tissues reflect the status of lipid peroxidation10. The SOD is an antioxidant enzyme catalyzes the conversion of superoxide anion into hydrogen peroxide which is transformed into water by a series of reaction catalyzed by catalase and glutathione peroxidase11. Oxidative stress biomarkers and lipid profiles have been presented as prognostic and diagnostic biomarkers for many animal diseases12-14. However, the investigation of these golden biomarkers in dairy cows at post-paturient period is not completely elucidated so far. Therefore, the current study aimed to evaluate potentials of oxidative stress biomarkers and lipid profile as diagnostic markers of ketosis in dairy cows at post-paturient period.


Animals: This study was carried out on a total number of 45 cows (3-9 weeks post-parturient), aging from 4-7 years old with average body weight of 650±15 kg from a private farm. The selected cows were assigned to two groups, first group represented control cows (n = 20) whereas second group (n = 25) consisted of ketotic cows. All cows were clinically examined every day until 4 weeks after parturition15. All applicable international, national and/or institutional guidelines for the care and use of animals were described.

Samples collection: Blood samples were collected from the jugular vein into plain tubes and were allowed to clot at room temperature. The harvested serum stored frozen at -20°C until the time of analysis of AST, GGT, glucose, total protein, albumin, NEFA, BHBA, total cholesterol, cholesterol ester, free cholesterol, TAG, MDA, NO, SOD and GSH. Urine samples have been collected from all animals for detection of ketonuria.

Biochemical analysis: The presence of ketone bodies in the urine was detected by commercial kits (Fujisawa pharmaceutical Co., Osaka, Japan). The levels of serum glucose, triglyceride, cholesterol as well as AST and GGT activities were determined in serum samples on a Beckman CX-7 auto-analyzer using the corresponding kits (Sigma Chemical Co., Ltd., Poole, Dorset, UK). Serum BHBA was determined by a kinetic enzymatic method using a commercially available kit (Ranbut D-3-hydroxybutyrate, Randox, Crumlin Co., Antrim, UK). Serum concentration of NEFA was carried out using commercially available test kits supplied by Randox laboratories Ltd.

Statistical analysis: All data was presented as mean±standard error of mean by using one way analysis of variance (ANOVA). All tests were performed using computer package of the statistical analysis system16.


Clinical examination: The diseased cows showed anorexia, ruminal stasis, constipation and significant reduction of milk production.

Profiles of proteins, lipids and enzymes: The profiles of proteins, lipids and enzymes showed in Table 1. The presented data indicated a significant increase (p≤0.05) in AST and GGT activities as well as concentrations of NEFA and BHBA in the serum of ketotic animals compare to the control. The data shown in the same Table 1 revealed a significant reduction (p≤0.05) in the values of glucose, total cholesterol, cholesterol ester, free cholesterol, TAG, total proteins, albumin and globulins in the serum of ketotic animals compare to the control.

Oxidative stress biomarkers: The values of oxidative stress biomarkers are illustrated in Table 2. The data summarized in this Table 2 revealed significant elevation of MDA and nitric oxide values and significant reduction in GSH concentration as well as SOD activity in the serum of ketotic animals compare to the control.

Table 1:Lipids, enzymes and protein profiles in serum of control and cows affected with ketosis
*Means are significantly different at the level (p≤0.05), AST: Aspartate aminotransferase, GGT: Gamma glutamyl transferase, TAG: Triacylglycerol, NEFA: Non-esterified free fatty acids, BHBA: β-hydroxylbutyric acid

Table 2:Oxidative stress biomarkers in serum of control and cows affected with ketosis
*Means are significantly different at the level (p≤0.05), MDA: Malonaldehyde, NO: Nitric oxide, SOD: Superoxide dismutase, GSH: Reduced glutathione

Table 3:
Spearmen’s correlation coefficient among traditional (BHBA and NEFA) and suggested (MDA, SOD, NO and GSH) biomarkers of ketosis in cows affected with ketosis
MDA: Malondialdehyde, SOD: Superoxide dismutase, NO: Nitric oxide, GSH: Reduced glutathione, BHBA: β-hydroxylbutyric acids, NEFA: Non-esterified free fatty acids

Correlation between traditional (BHBA and NEFA) and suggested (MDA, SOD, NO and GSH) biomarkers of ketosis in cows affected with ketosis: The data summarized in Table 3 indicated that, BHBA was positively correlated with NEFA (r = 0.974, p = 0.000). Furtherly, both BHBA and NEFA were inversely correlated with SOD (r = -0.933 and -0.908) and GSH (r = -0.894 and -0.910), respectively. In addition, MDA was inversely correlated with SOD (r = -0.899) and GSH (r = -0.886) and positively correlated with NO (r = 0.952), BHBA (r = 0.955) and NEFA (r = 0.963).


The occurrence of ketosis in cows at post-parturient phase perhaps owed to lack of dry matter intake around parturition, increase demands for glucose and insufficient propionate production17,18. Ketotic animals have been diagnosed by positive findings of clinical examination and confirmed by positive ketone bodies test in the urine. The clinical signs observed in the ketotic cows were the same observed in previous study on cows19 and buffaloes20,21. Beside the clinical signs that observed in ketotic cows of the current study, ketosis has been approved by positive test of ketone bodies in urine and ketonuria. In addition, the significant increase of NEFA and BHBA in the serum of ketotic cows at post-parturient period confirmed the observed clinical findings. The significant increase of AST and GGT activities in the serum of ketotic cows compared to control as observed in the current study indicated liver dysfunction22,23. The release of liver enzymes (AST and GGT) may attribute to increased hepatic cell membrane permeability as a result of infiltration of hepatic cells with fat24. The hepatic dysfunction in ketotic cows has been confirmed also by observed low TAG level along with high AST and GGT activities compare to control animals. In addition, the significant decrease in total protein and albumin level in ketotic animals of the current study compare to control indicated liver dysfunction25. The significant reduction of cholesterol levels in ketotic cows compared with normal ones as reported in the current study may attribute to liver dysfunction which reduces cholesterol biosynthesis26. Similar findings have been observed in cattle27 and buffaloes20. In the contrast other study reported significant increase in cholesterol levels in ketotic animals28. The significant decrease in cholesterol ester in ketotic cows may attribute to the negative effect of postpaturient ketosis on synthesis of LCAT, an enzyme responsible for formation of cholesterol ester from peripheral cholesterol20,29. Similar results have been obtained in ketotic cows and bufflaoes20,25. The significant reduction in glucose level and higher BHBA in ketotic cows has been reported earlier in cows30-32 and buffaloes20,33. Hypoglycemia may occur due to imbalance between glucose intake34 and glucose utilization in the mammary gland during lactation period postpartum31. As a response to low blood glucose level, fat mobilization is initiated7,35 and subsequent elevation of NEFA oxidation and production of BHBA has been occurred to compensate the energy loss as a result of absence of glucose36. However, as a result of elevated rate of fatty acid oxidation, free radicals have been produced causing lipid peroxidation and oxidative stress. In addition the formed ketone bodies (BHBA) considered as important source of free radicals and initiation of oxidative stress. Therefore, in the current study, MDA level has been increased significantly in cows affected with ketosis compared to control. Similar findings have been reported in cows37, buffaloes20,21, human38 and rabbits39. In the current study NO level has been elevated in in cows affected with ketosis compared to control. It has been postulated that ketosis enhanced the NO production40. The significant positive correlation between MDA and BHBA and the negative correlation between MDA as well as BHBA and NO have been observed earlier in buffaloes21. The negative correlation among MDA, NEFA as well as BHBA and SOD and GSH was reported for first time in cows affected with ketosis in the current study.


At post-paturient period in cows, blood glucose level decreased, lipid mobilization and fatty acid oxidation increased with subsequent increase in ketone bodies (BHBA) which creating a state of lipid peroxidation (MDA) and oxidative stress. The enzymatic (SOD) and non-enzymatic (GSH) antioxidants have been depleted as a trial to counteract the stressful situation.


Oxidative stress biomarkers and lipid profiles could be used as promising biomarkers for ketosis in dairy cows at post-paturient period. The antioxidant therapy may useful in the treatment of ketosis in cows at post-paturient period.


Authors thank Deanship of Scientific Research, KFU, Saudi Arabia for financial support.

Akamatsu, H., Y. Saitoh, M. Serizawa, K. Miyake, Y. Ohba and K. Nakashima, 2007. Changes of serum 3-methylhistidine concentration and energy-associated metabolites in dairy cows with ketosis. J. Vet. Med. Sci., 69: 1091-1093.
CrossRef  |  PubMed  |  Direct Link  |  

Anantwar, L.G. and B. Singh, 1993. Epidemiology, clinico-pathology and treatment of clinical ketosis in buffalos (Bubalus bubalis). Indian Vet. J., 70: 152-156.
Direct Link  |  

Bobe, G., J.W. Young and D.C. Beitz, 2004. Pathology, etiology, prevention and treatment of fatty liver in dairy cows. J. Dairy Sci., 87: 3105-3124.
CrossRef  |  PubMed  |  Direct Link  |  

Bremmer, D.R., S.L. Trower, S.J. Bertics, S.A. Besong, U. Bemabucci and R.R.Grummer, 2000. Etiology of fatty liver in dairy cattle: Effects of nutritional and hormonal status on hepatic microsomal triglyceride transfer protein. J. Dairy Sci., 83: 2239-2251.
CrossRef  |  PubMed  |  Direct Link  |  

Cebra, C.K., F.B. Garry, D.M. Getz and M.J. Fettman, 1997. Hepatic lipidosis in anorectic, lactating Holstein cattle: A retrospective study of serum biochemical abnormalities. J. Vet. Int. Med., 11: 231-237.
CrossRef  |  PubMed  |  Direct Link  |  

Celik, S. and H. Karagul, 2005. The red blood cell membrane proteins in rabbits with experimental ketosis. Turk. J. Vet. Anim. Sci., 29: 151-155.
Direct Link  |  

Contreras, G.A. and L.M. Sordillo, 2011. Lipid mobilization and inflammatory responses during the transition period of dairy cows. Comp. Immunol. Microbiol. Infect. Dis., 34: 281-289.
CrossRef  |  Direct Link  |  

Dann, H.M. and J.K. Drackley, 2005. Carnitine palmitoyltransferase i in liver of periparturient dairy cows: Effects of prepartum intake, postpartum induction of ketosis and periparturient disorders. J. Dairy Sci., 88: 3851-3859.
CrossRef  |  PubMed  |  Direct Link  |  

Drackley, J.K., 1999. Biology of dairy cows during the transition period: The final frontier? J. Dairy Sci., 82: 2259-2273.
CrossRef  |  PubMed  |  Direct Link  |  

Drackley, J.K., H.M. Dann, G.N. Douglas, N.A.J. Guretzky, N.B. Litherland, J.P. Underwood and J.J. Loor, 2005. Physiological and pathological adaptations in dairy cows that may increase susceptibility to periparturient diseases and disorders. Ital. J. Anim. Sci., 4: 323-344.
CrossRef  |  Direct Link  |  

Duffield, T., 2000. Subclinical ketosis in lactating dairy cattle. Vet. Clin. North Am. Food Anim. Pract., 16: 231-253.
PubMed  |  Direct Link  |  

El-Deeb, W.M. and S.M. El-Bahr, 2010. Investigation of selected biochemical indicators of Equine Rhabdomyolysis in Arabian horses: Pro-inflammatory cytokines and oxidative stress markers. Veterinary Res. Commun., 34: 677-689.
CrossRef  |  Direct Link  |  

El-Deeb, W.M. and S.M. El-Bahr, 2014. Acute-phase proteins and oxidative stress biomarkers in water buffalo calves subjected to transportation stress. Comp. Clin. Pathol., 23: 577-582.
CrossRef  |  Direct Link  |  

El-Deeb, W.M., T.A. Fouda and S.M. El-Bahr, 2014. Clinico-biochemical investigation of paratuberculosis of dromedary camels in Saudi Arabia: Proinflammatory cytokines, acute phase proteins and oxidative stress biomarkers. Pak. Vet. J., 34: 484-488.
Direct Link  |  

Ghanem, M.M. and W.M. El-Deeb, 2010. Lecithin Cholesterol Acyltransferase (LCAT) activity as a predictor for ketosis and parturient haemoglobinuria in Egyptian water buffaloes. Res. Vet. Sci., 88: 20-25.
CrossRef  |  PubMed  |  Direct Link  |  

Goff, J.P. and R.L. Horst, 1997. Physiological changes at parturition and their relationship to metabolic disorders. J. Dairy Sci., 80: 1260-1268.
CrossRef  |  PubMed  |  Direct Link  |  

Gonzalez, F.D., R. Muino, V. Pereira, R. Campos and J.L. Benedito, 2011. Relationship among blood indicators of lipomobilization and hepatic function during early lactation in high-yielding dairy cows. J. Vet. Sci., 12: 251-255.
CrossRef  |  Direct Link  |  

Grummer, R.R., 1995. Impact of changes in organic nutrient metabolism on feeding the transition dairy cow. Anim. Sci., 73: 2820-2833.
CrossRef  |  PubMed  |  Direct Link  |  

Jain, S.K., R. Mcvie, R. Jackson, S.N. Levine and G. Lim, 1999. Effect of hyperketonemia on plasma lipid peroxidation levels in diabetic patients. Diabetes Care, 22: 1171-1175.
CrossRef  |  Direct Link  |  

Jonsson, N.N., M.R.S. Fortes, E.K. Piper, D.M. Vankan, J.P.J. de Cisneros and T. Wittek, 2013. Comparison of metabolic, hematological and peripheral blood leukocyte cytokine profiles of dairy cows and heifers during the periparturient period. J. Dairy Sci., 96: 2283-2292.
CrossRef  |  Direct Link  |  

Jorritsma, R., H. Jorritsma, Y.H. Schukken, P.C. Bartlet, T. Wensing and G.H. Wentink, 2001. Prevalence and indicators of post partum fatty infiltration of the liver in nine commercial dairy herds in The Netherlands. Livest. Prod. Sci., 68: 53-60.
CrossRef  |  Direct Link  |  

Karsai, F. and M. Schafer, 1984. Diagnostic experiences with metabolic liver diseases of dairy cows. Monta. Fur. Vet., 39: 181-186.

Kelton, D.F., K.D. Lissemore and R.E. Martin, 1998. Recommendations for recording and calculating the incidence of selected clinical diseases of dairy cattle. J. Dairy Sci., 81: 2502-2509.
CrossRef  |  Direct Link  |  

Marcos, E., A. Mazur, P. Cardot and Y. Rayssiguier, 1990. Serum apolipoproteins B and A-I and naturally occurring fatty liver in dairy cows. Lipids, 25: 575-577.
CrossRef  |  Direct Link  |  

Melendez, P., M.P. Marin, J. Robles, C. Rios, M. Duchens and L. Archbald, 2009. Relationship between serum nonesterified fatty acids at calving and the incidence of periparturient diseases in Holstein dairy cows. Theriogenology, 72: 826-833.
CrossRef  |  Direct Link  |  

Miller, J.K., E. Brzezinska-Slebodzinska and F.C. Madsen, 1993. Oxidative stress, antioxidants and animal function. J. Dairy Sci., 76: 2812-2823.
CrossRef  |  PubMed  |  Direct Link  |  

Nakagawa, H. and N. Katoh, 1998. Reduced activity of lecithin: Cholesterol acyltransferase in the serum of cows with ketosis and left displacement of the abomasum. Vet. Res. Commun., 22: 517-524.
CrossRef  |  Direct Link  |  

Nazifi, S., M.M. Fani, E. Rowghani and M.R. Behbood, 2008. Studies on the relationship between sub-clinical ketosis and liver injuries within the first two months of lactation period in high producing. Int. J. Dairy. Sci., 3: 29-35.
CrossRef  |  Direct Link  |  

Padilla, L., K. Shibano, J. Inoue, T. Matsui and H. Yano, 2005. Plasma vitamin c concentration is not related to the incidence of ketosis in dairy cows during the early lactation period. J. Vet. Med. Sci., 76: 883-886.
Direct Link  |  

Radostits, O.M., C.C. Gay, K.W. Hinchcliff and P.D. Constable, 2007. Veterinary Medicine: A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats. 10th Edn., Saunders Elsevier, Philadelphia, PA USA.

Rahman, I., S.K. Biswas and A. Kode, 2006. Oxidant and antioxidant balance in the airways and airway diseases. Eur. J. Pharmacol., 533: 222-239.
CrossRef  |  PubMed  |  Direct Link  |  

SAS., 2002. Statistical Analysis System. 1st Edn., SAS Institute Inc., Cary, NC., USA.

Sharma, I.J. and R. Kumar, 2001. Correlation of some blood biochemicals with ketone bodies in normal and sub-clinical ketotic bovines. Indian J. Anim. Sci., 71: 1029-1031.
Direct Link  |  

Steen, A., H. Gronstol and P.A. Torjensen, 1997. Glucose and insulin responses to glucagon injection in dairy cows with ketosis and fatty liver. J. Vet. Med. A, 44: 521-530.
PubMed  |  Direct Link  |  

Tothova, C., O. Nagy and G. Kovac, 2014. Relationship between some variables of protein profile and indicators of lipomobilization in dairy cows after calving. Archiv Tierzucht, 57: 1-9.
Direct Link  |  

West, H.J., 1990. Effect on liver function of acetonaemia and the fat cow syndrome in cattle. Res. Vet. Sci., 48: 221-227.
PubMed  |  Direct Link  |  

Yameogo, N., G.A. Ouedraogo, C. Kanyandekwe and G.J. Sawadogo, 2008. Relationship between ketosis and dairy cow's blood metabolites in intensive production farms of the periurban area of Dakar. Trop. Anim. Health Prod., 40: 483-490.
CrossRef  |  Direct Link  |  

Yousef, M.I., A.A. Saad and L.K. El-Shennawy, 2009. Protective effect of grape seed proanthocyanidin extract against oxidative stress induced by cisplatin in rats. Food Chem. Toxicol., 47: 1176-1183.
CrossRef  |  Direct Link  |  

Youssef, M.A., S.A. El-Khodery, W.M. El-Deeb and W.E. Abou Elamiem, 2010. Ketosis in buffalo (Bubalus bubalis): Clinical findings and the associated oxidative stress level. Trop. Anim. Health Product., 42: 1771-1777.
CrossRef  |  PubMed  |  Direct Link  |  

Zdzisinska, B., J. Filar, R. Paduch, J. Kaczor, I. Lokaj and M. Kandefer-Szerszen, 2000. The influence of ketone bodies and glucose on Interferon, tumor necrosis factor production and NO release in bovine aorta endothelial cells. Vet. Immunol. Immunopathol., 74: 237-247.
CrossRef  |  Direct Link  |  

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