Prevalence and biochemical alterations of subclinical ketosis in lactating buffaloes in Bidar

Introduction

Indian buffaloes are important source of milk supply today and yield nearly three times as much milk as cows (Yadav et al., 2017) [1] . Among various production diseases, ketosis is an important production disease and continues to cause significant economic losses to the farmers. Subclinical ketosis (SCK) causes greater losses than clinical ketosis because it occurs more frequently and often cannot be detected by farmers (Brunner et al., 2019) [2] . As the course of the diseases is often subclinical and commonly goes unnoticed by the farmers, early detection is very important to mitigate further losses (Enjalbert et al., 2001) [3] . Diagnosis of individual cases of subclinical ketosis at an earlier point in the course of the disease would permit earlier treatment and help to extenuate further losses. Due to the absence of signs or symptoms for subclinical ketosis, blood, urine and milk tests are used as diagnostic tools to detect ketone bodies concentration in circulation. The volatile nature of acetone and relatively unstable nature acetoacetate make them unreliable as a cow side test. Beta hydroxy butyric acid (BHBA) on the other hand is more stable and is detectable in urine, milk and blood.

Material and methods

A total of 108 early lactating buffaloes maintained in various organized and unorganized dairy farms in and around Bidar were screened for subclinical ketosis. To diagnose the subclinical ketosis in buffaloes, pen side diagnostic tests was employed viz., enzyme based electrochemical test for blood BHBA using Precision Xtra® blood glucose and ketone monitoring system, the drop of blood was placed on the BHBA strip and results were recorded. The blood samples were collected from six subclinical ketosis affected buffaloes and separated serum for estimation of serum Glucose, AST, Total protein, Albumin, Calcium and Phosphorus by using Semi-automated clinical chemistry analyzer. A total of six apparently healthy early lactating buffaloes which were negative for subclinical ketosis were keptAs healthy control group.

Results and discussion

In the present investigation, a total of 108 apparently healthy buffaloes suspected of subclinical ketosis were screened using a handheld ketone meter (Precision Xtra®), employing a cutoff point of ≥ 1.0 mmol/L of β-hydroxybutyrate (BHBA) (Whitaker et al., 1983) [4] . Out of these, 16 buffaloes were found positive, recording an overall prevalence of 14.81 per cent. Similar prevalence rates were also reported by Katta (2003) [5] , Padmaja (2009) [6] , and Youssef et al. (2010) [7] . However, Surender (2018) [8] reported comparatively lower prevalence of subclinical ketosis in recently calved buffaloes. Earlier reports have also documented varying rates of prevalence of subclinical ketosis in buffaloes in India (Gupta, 2012 [9] ; Krishna et al., 2014 [10] ). Regional and country-wise differences in animal husbandry practices play an important role in ketosis prevalence. Differences in study design and methodology may also account for variation in prevalence estimates. Such wide variation in prevalence might be attributed to agro-climatic conditions, climatic changes, breed susceptibility, stage of lactation, production potential, feeding habits, and managemental practices followed by owners. In the present investigation, the highest prevalence of subclinical ketosis in buffaloes was recorded in the third parity, followed by fourth parity, second parity, and fifth and above parity, with the lowest prevalence observed in first parity animals. These results were in agreement with McArt et al. (2012) [11] , who reported that the prevalence of subclinical ketosis was highest during the third parity, followed by the first parity. Similarly, Krishna et al. (2014) [10] recorded the highest prevalence of SCK during the fourth lactation, followed by third and second lactations, with the lowest prevalence during the first lactation. Kumar et al. (2015) [12] reported that most cases of ketosis occurred during the third lactation in animals aged 3-9 years. Vanholder et al. (2015) [13] reported that the risk of developing subclinical ketosis was higher in second parity and older cows compared with heifers. The higher prevalence of SCK in the third parity might be due to the fact that animals at this stage attain their genetically determined peak productivity and are unable to withstand the metabolic shift arising from increased nutritional demands, making them more susceptible to production diseases (Thirunavukkarasu et al., 2010) [14] . The mean BHBA concentration (mmol/L) in the SCKaffected group was significantly higher compared to the healthy control group. The findings of the present study are in accordance with observations reported by McArt et al. (2012) [11] , Ghanem et al. (2016) [15] , and Surender (2018) [8] . BHBA is regarded as the gold standard for diagnosing subclinical ketosis in cows and buffaloes during lactation, as it is more stable than acetoacetate and acetone. BHBA is the predominant circulating ketone body and remains relatively stable in whole blood, plasma, and serum (Dohoo and Martin, 1984) [16] . Serum glucose level is considered one of the key indicators of energy status in ruminants. In the present study, blood glucose concentration was significantly lower in subclinical ketosisaffected buffaloes compared to the healthy control group. These results correlate well with the findings of Youssef et al. (2010) [7] , Padmaja and Rao (2013) [17] , and Surender (2018) [8] in subclinical ketotic buffaloes, as well as Hagawane et al. (2009) [18] in early lactation buffaloes, and Youssef et al. (2010) [7] and Bali et al. (2016) [19] in clinically ketotic buffaloes. The reduced blood glucose concentration during early lactation is attributed to the large withdrawal of glucose by the mammary gland for lactose synthesis (Schultz, 1968) [20] . Hypoglycaemia may also occur due to intake of lowenergy diets, particularly during early lactation when glucose demand by the mammary gland is high (Nazifi et al., 2008) [21] . Glucose serves as the principal substrate for lactose synthesis, and approximately 60-80 per cent of circulating glucose is utilized by the mammary gland for milk production (Annison and Linzell, 1963) [22] . Consequently, hypoglycaemia observed in early lactating buffaloes in the present study can be attributed to increased glucose utilization for lactose synthesis. Aspartate transaminase (AST) activity showed a significant increase in subclinical ketosis-affected buffaloes compared with healthy controls. These findings were in agreement with Youssef et al. (2010) [7] , Padmaja and Rao (2013) [17] , and Bali et al. (2016) [19] . However, Surender (2018) [8] recorded a non-significant increase in AST levels in subclinical ketotic buffaloes compared with healthy animals. Furthermore, Ghanem et al. (2016) [15] observed a significantly higher AST activity in clinically ketotic cows compared to SCK and healthy cows. Aspartate transaminase is a non-specific liver enzyme, and its increased activity in dairy animals is often associated with fatty liver syndrome (Djokovic et al., 2016) [23] . Serum AST activity correlates with the degree of hepatic fatty infiltration (Dokovic et al., 2012) [24] . During early lactation, cows experiencing severe negative energy balance (NEB) exhibit reduced hepatic capacity to export triglycerides (TAG) as very-low-density lipoproteins (VLDL), leading to hepatic accumulation of TAG and subsequent fatty liver development (Rukkwamsuk et al., 1999) [25] , ketosis (Herdt, 2000) [26] , and increased hepatocellular membrane permeability with subsequent release of AST (Karasai and Schefar, 1984) [27] . Therefore, elevated AST levels observed in subclinical ketotic buffaloes may be attributed to negative energy balance. The present study revealed no significant difference in total protein levels between healthy control and SCK-affected groups. This finding corroborates reports by Youssef et al. (2010) [7] , Hussein et al. (2015) [28] , and Surender (2018) [8] . However, Padmaja and Rao (2013) [17] and Singh (2015) [29] reported significantly lower total plasma protein levels in SCK-affected buffaloes. Similarly, Singh et al. (2017) [30] and Paramesh et al. (2020) [31] reported a significant decrease in serum total protein levels in subclinical ketotic cows. In contrast, Hagawane et al. (2009) [18] reported slightly elevated total protein levels in early lactation buffaloes. Total protein concentration provides information regarding liver and kidney function as well as nutritional status (Stojevic et al., 2005) [32] . The absence of significant differences in total protein levels between groups in the present study indicates no derangement in serum total protein concentration in subclinical ketosis. In the present study, a significant decrease in albumin levels was observed in the SCK-affected group compared with healthy controls. Similar findings were reported by Padmaja and Rao (2013) [17] and Singh (2015) [29] . These results were also in agreement with Reddy (2015) [33] , who reported significantly lower albumin concentrations in SCK-affected dairy cows. However, Youssef et al. (2010) [7] and Hussein et al. (2015) [28] reported non-significant differences in albumin levels in subclinical ketotic buffaloes, while Akgul et al. (2017) [34] and Singh et al. (2017) [30] reported no significant difference in albumin levels in subclinical ketotic cows. level in SCK affected dairy cows.The decrease in albumin The decreased albumin level in SCK-affected buffaloes could be attributed to hepatic insufficiency. Albumin is an indicator of liver synthetic function (West, 1990) [35] , and a decreased concentration of albumin reflects hepatic insufficiency (Whitaker, 2000) [36] . The decreased liver synthesis of albumin may be induced by the development of fatty infiltration of the liver (Lubojacka et al., 2005) [37] . The serum calcium level was found to be low in the affected group, indicating hypocalcaemia. These findings were in accordance with those reported by Hagawane et al. (2009) [18] , Padmaja and Rao (2013) [17] , and Singh (2015) [29] in subclinical ketotic buffaloes, and Bali et al. (2016) [19] in clinically ketotic buffaloes. However, Youssef et al. (2010) [7] found nonsignificant changes in calcium levels in post-parturient ketotic and subclinical ketotic buffaloes. This could be attributed to increased loss of base in urine to compensate for ketosisinduced acidosis.The lower level of serum calcium in subclinical ketosis can also be attributed to a high concentration of β-hydroxybutyrate (BHBA), which impairs the absorption and utilization of calcium in dairy cows during the early lactation period (Zhang et al., 2009) [38] . Consequently, the decrease in calcium levels could be a result of impaired absorption of food metabolites from the gastrointestinal tract, excessive losses through urine and colostrum (especially during excessive milking), and insufficient mobilization from the skeleton (Hagawane et al., 2009) [18] . In the present study, a significantly low level of phosphorus was recorded in the affected group. This finding is in accordance with Youssef et al. (2010) [7] and Hagawane et al. (2009) [18] , who reported significantly low phosphorus levels in subclinical ketotic buffaloes. Moreover, Bali et al. (2016) [19] reported significantly low phosphorus levels in clinically ketotic buffaloes, and Ghanem et al. (2016) [15] demonstrated a significant decrease in serum phosphorus levels in ketotic cows. The decrease in serum phosphorus level observed in the present study might be due to the large amount of inorganic phosphorus drained from blood for milk production, hyperparathyroidism induced by low serum calcium levels, and increased urinary losses (Valk et al., 2002) [39] . The lower level of serum phosphorus in ketosis may also be attributed to compensation for ketosis-induced acidosis, which triggers increased excretion of phosphorus via urine (Fatur, 1994) [40] .

Conclusion

Hand held ketone meter Precision Xtra® was most specific cow side diagnostic testto detectsubclinical ketosis in lactating buffaloes. Overall prevalence of Subclinical ketosis inlactating buffaloes was 14.81 per cent.Results of biochemical study indicated statistically significant decrease in serumglucose, albumin, serum calcium and serum phosphorus, further statistically significant increase in AST in Subclinical ketosis affected buffaloes.

References

1. Yadav AK, Singh J, Yadav SK. Characteristic features of registered indigenous buffalo breeds of India: a review. Int J Pure Appl Biosci. 2017;5(4):825-831.
2. Brunner N, Groeger S, Raposo CJ, Bruckmaier RM, Gross JJ. Prevalence of subclinical ketosis and production diseases in dairy cows in Central and South America, Africa, Asia, Australia, New Zealand, and Eastern Europe. Transl Anim Sci. 2019;3(1):84-92.
3. Enjalbert F, Nicot MC, Bayourthe C, Moncoulon R. Ketone bodies in milk and blood of dairy cows: relationship between concentrations and utilization for detection of subclinical ketosis. J Dairy Sci. 2001;84:583589.
4. Whitaker DA, Kelly JM, Smith EJ. Subclinical ketosis and serum beta-hydroxybutyrate levels in dairy cattle. Br Vet J. 1983;139(5):462-463.
5. Katta A. Studies on subclinical ketosis in buffaloes. M.V.Sc. Thesis. Tirupati: Sri Venkateswara Veterinary University; 2003.
6. Padmaja K. Clinical studies on postparturient indigestion with particular reference to hepatic disorders and production diseases in buffaloes. Ph.D. Thesis. Tirupati: Sri Venkateswara Veterinary University; 2009.
7. Youssef MA, El-Khodery SA, El-Deeb WM, El-Amaiem WEA. Ketosis in buffalo (Bubalus bubalis): clinical findings and the associated oxidative stress level. Trop Anim Health Prod. 2010;42(8):1771-1777.
8. Surender. Subclinical ketosis in buffaloes and its therapeutic management. M.V.Sc. Thesis. Telangana: P.V. Narsimha Rao Veterinary University; 2018.
9. Gupta M. Epidemiological and therapeutic studies of subclinical ketosis in lactating buffaloes. M.V.Sc. Thesis. Jabalpur: Nanaji Deshmukh Veterinary Science University; 2012.
10. Krishna KS, Suresh K, Sreenu M, Sundar NS. Prevalence of subclinical hypocalcaemia and subclinical ketosis in buffaloes. Buffalo Bull. 2014;33(1):107-110.
11. McArt JAA, Nydam DV, Oetzel GR. Epidemiology of subclinical ketosis in early lactation dairy cattle. J Dairy Sci. 2012;95(9):5056-5066.
12. Kumar A, Kumar T, Kumar P, Charaya G, Sindhu N, Kumari S, et al. Hemato-biochemical studies on clinical cases of primary ketosis in buffaloes. J Anim Res. 2015;5(3):443-448.
13. Vanholder T, Papen J, Bemers R, Vertenten G, Berge ACB. Risk factors for subclinical and clinical ketosis and association with production parameters in dairy cows in the Netherlands. J Dairy Sci. 2015;98(2):880-888.
14. Thirunavukkarasu M, Kathiravan G, Kalaikannan A, Jebarani W. Prevalence of ketosis in dairy farms: a survey in Tamil Nadu. Tamil Nadu J Vet Anim Sci. 2010;6(4):193-195.
15. Ghanem MM, Mahmoud ME, Abd El-Raof YM, El-Attar HM. Alterations in biochemical parameters and hepatic ultrasonography with reference to oxidant injury in ketotic dairy cows. Benha Vet Med J. 2016;31(2):231240.
16. Dohoo IR, Martin SW. Subclinical ketosis: prevalence and associations with production and disease. Can J Comp Med. 1984;48(1):1-5.
17. Padmaja K, Rao DST. Clinical, biochemical and therapeutic responses of Laura bolin and Ketonex to postparturient indigestion related subclinical ketosis in buffalo. Anim Sci Rep. 2013;7(1):35-40.
18. Hagawane SD, Shinde SB, Rajguru DN. Haematological and blood biochemical profile in lactating buffaloes in and around Parbhani city. Vet World. 2009;2(12):467469.
19. Bali G, Hussain K, Razzaque WAA, Sharma U, Beigh SA. Clinico-biochemical studies of ketosis in buffalo (Bubalus bubalis). Buffalo Bull. 2016;35(1):27-32.
20. Schultz LH. Ketosis in dairy cows. J Dairy Sci. 1968;51:1133-1140.
21. Nazifi S, Mohebbi FM, Rowghani E, Behbood MR. Studies on the relationship between subclinical ketosis and liver injuries within the first two months of lactation period in high producing Iranian Holstein cows. Int J Dairy Sci. 2008;3(1):29-35.
22. Annison EF, Linzell JL. Oxidation of glucose and acetate by the mammary gland. J Physiol. 1963;185:372-355.
23. Djokovic R, Ilic Z, Kurcubic V, Petrovic M, Cincovic M, Petrovic MP, et al. Diagnosis of subclinical ketosis in dairy cows. Biotechnol Anim Husb. 2019;35(2):111-125.
24. Dokovic R, Ilic Z, Kurcubic V, Petrovic MP, CaroPetrovic V, Milosevic B, et al. Determination of metabolic and nutritional status in dairy cows during early and mid lactation. Biotechnol Anim Husb. 2016;32(1):1-8.
25. Rukkwamsuk T, Kruip TAM, Wensing T. Relationship between overfeeding and overconditioning in the dry period and the problems of high producing dairy cows during the postparturient period. Vet Q. 1999;21(3):7177.
26. Herdt TH. Ruminant adaptation to negative energy balance: influences on the etiology of ketosis and fatty liver. Vet Clin North Am Food Anim Pract. 2000;16(2):215-230.
27. Karasai F, Schefar M. Diagnostic experiences with metabolic liver diseases of dairy cows. Monatsh Veterinarmed. 1984;39:181-186.
28. Hussein HA, Abdel-Raheem SM, Abd-Allah M, Senosy W. Effects of propylene glycol on the metabolic status and milk production of dairy buffaloes. Tierarztl Prax Ausg G Grosstiere Nutztiere. 2015;43(1):25-34.
29. Singh R. Prediction and management of metabolic diseases in dairy cows and buffaloes. Ph.D. Thesis. Ludhiana: Guru Angad Dev Veterinary and Animal Sciences University; 2015.
30. Singh G, Randhawa SS, Uppal SK, Randhawa CS, Chand N. Evaluation of efficacy of propylene glycol in the treatment of subclinical ketosis and its effect on plasma concentration of various metabolic parameters. J Anim Res. 2017;7(4):691-697.
31. Paramesh SC, Kumar MA, Ramesh PT, Upendra HA, Narayanaswamy M, Manjunath KP. Detection of subclinical ketosis by hand-held ketometer. Pharm Innov J. 2020;9(2):391-393.
32. Stojevic Z, Pirsljin J, Milinkovic-Tur S, Zdelar-Tuk M, Ljubic BB. Activities of AST, ALT and GGT in clinically healthy dairy cows during lactation and in the dry period. Vet Arh. 2005;75(1):67-73.
33. Reddy S. Studies on subclinical ketosis in cows and its therapeutic management. M.V.Sc. Thesis. Telangana: P.V. Narsimha Rao Veterinary University; 2015.
34. Akgul G, Şenturk S, Mecitoglu Z, Akkaya F. Relationship between some biochemical parameters in healthy and dairy cows with subclinical ketosis. Veterinaria. 2017;66(2):83-86.
35. West HJ. Effect on liver function of acetonaemia and the fat cow syndrome in cattle. Res Vet Sci. 1990;48(2):221227.
36. Whitaker DA. Use and interpretation of metabolic profiles. In: The health of dairy cattle. Oxford: Blackwell Scientific Publications; 2000. p. 89-93.
37. Lubojacka V, Pechova A, Dvorak R, Drastich P, Kummer V, Poul J. Liver steatosis following supplementation with fat in dairy cow diets. Acta Vet Brno. 2005;74(2):217-224.
38. Zhang Z, Liu G, Li X, Wang Z, Kong T, Zhang N, et al. β-hydroxybutyrate, glucose, calcium, phosphorus and vitamin C concentrations in blood of dairy cows with subclinical ketosis during early lactation. Bull Vet Inst Pulawy. 2009;53:71-74.
39. Valk H, Sebek LBJ, Beynen AC. Influence of phosphorus intake on excretion and blood plasma and saliva concentrations of phosphorus in dairy cows. J Dairy Sci. 2002;85(10):2642-2649.
40. Fatur B. Ca, P, Mg, K and Na serum levels of dairy cows with subclinical ketosis. In: Proceedings of the 18th World Buiatrics Congress; 1994; Bologna, Italy. Bologna: Societa Italiana di Buiatria; 1994. p. 12571260.