BHB METER AS DIAGNOSTIC KIT FOR DIAGNOSING SUBCLINICAL KETOSIS IN DAIRY COWS
One of the most important problems associated with postpartum cows is the negative energy balance (NEB) resulting in a metabolic condition termed as ketosis (Gross and Bruckmaier, 2019; Overton et al., 2017). The sudden change in the metabolic status during the pregnancy to lactation transition and deficient feed intake leads to the NEB in cows that are in early lactation or in postpartum period which has become a serious issue in milking industry (Gohary et al., 2016; Mostert et al., 2018; Mozduri et al., 2018). Moreover, the asymptomatic nature of the condition, reduced milk yields with sudden energy loss and associated sickness make the cows more susceptible to infections and poor reproductive performance in the later stages (Pascottini and LeBlanc, 2020; Raboisson et al., 2014; Xu et al., 2018). Unnoticed or poor management during the early days of lactation reduces the milking efficiency that directly leads to production loss that impacts economic growth (Mostert et al., 2018; Raboisson et al., 2015). Moreover, NEB impact the reproductive ability of the cows that in the recovery stage after parturition that predispose animals to acute and recurrent inflammatory attacks. Major indications of NEB are drop in milk yield, reduction in body weight, inappetence with occasional neurological signs and altered blood levels of β-hydroxybutyrate (BHB), a short chain fattry acid that belong to the group, non-esterified fatty acid (NEFA), gluconeogenic precursors, rumen-choline level and somatotropins (Ceciliani et al., 2018; Shahzad et al., 2019). In general, the increase in blood BHB leading to acetonemia in severe ketosis condition is classified as clinical ketosis. While the increase in blood BHB without clinical signs of illness is termed as subclinical ketosis. Nevertheless, financial damages due to subclinical ketosis or NEB can be minimized by constant or early detection and treatment of cows in the lactation period. Among the various cellular indicators that are altered, the thresholds of ketone bodies such as BHB, acetoacetate (AA), and acetone (Ac) has been used for the identification of clinical condition and its severity. Clinical ketosis is well defined as having BHB blood level of ≥2.5 mmol/l (26.2 mg/dl) and generally affects up to 15% of cows, whereas sub-clinical ketosis begins at ≥1.2 mmol/L (12.4 mg/dl), and shows a prevalence of over 40% of cows in contemporary commercial herds. Apparently normal cows had about 150 µmol/L average concentrations of BHB in the milk which was about ten times lower than blood concentrations. These levels increased up to 1mmol/L in clinical ketosis (Benedet et al., 2019; Jezek et al., 2017). The statistically profound positive equavalence between the concentration of BHB in blood and milk was found during NEB or ketosis. Hence, identification of milk ketosis was found to be relevant and encouraging because of its ease of sampling and the possibility for routine monitoring without any invasive method. Existing methods for bovine ketosis diagnosis depend on the availability of testing kits and the affordability of the animal owners. For instance, test kits that are in use are Rothera’s powder, ketone meters, acetone or acetoacetate test strips, colorimetric and spectrophotometric methods (Oetzel, 2004; Overton et al., 2017). Moreover, researchers have also developed testing methods with super-specific identification of molecules using quantum dots phosphor particles (Weng et al., 2015a), Fourier-transform infrared spectroscopy (FTIR) based methods (Pralle et al., 2018), Nuclear magnetic resonance (NMR) (Basoglu et al., 2020) and microfluidics technologies (Weng et al., 2015b). Despite demonstrable diagnostic accuracy, drawbacks such as high costs, high turnaround times, and requirement of expertise make them less attractive for field applications. Hence, in order to develop a portable a milk-based ketosis diagnosis method has been developed based on colorimetric detection that demonstrated the limit of detection (LOD) in the milk samples equivalent to commercial kits. The developed kit substantiated with good sensitivity in diagnosing complex milk samples, and this platform can be used in testing other body fluid that are of clinical interest. The developed method provides a instant, sensuous and assured tool for the continual observation of cows for subclinical ketosis as a point-of-care (POC) assay.
Advances in genetic techniques has resulted in improvement in dairy cows markedly increasing milk yield over the last three decades. But this increased milk production is often associated with poor reproductive performance and other metabolic disorders. Transition period as described by Grummer, (1995) is the most vulnerable and critical period experienced by cow that extends from three weeks prepartum to three weeks postpartum. During this period cow undergoes major physiological, nutritional, immunological, endocrine and metabolic changes that are necessary for the cow to shift from gestational non-lactating stage to parturition and lactogenesis. Hayirli et al. (2002) reported one-third decrease in feed intake experienced by the cow during last three weeks prior to calving and significant reduction in final week before parturition. This is mainly due to less capacity for rumen to expand because of increased size of foetus and high concentrations on estrogens levels. After parturition there is increase in demand for nutrient and energy for lactation but the inability of the cow to cope with the increasing energy demands due to decreased DMI results in negative energy balance. To meet the energy requirements depletion of body fat stores occurs. These processes are accompanied by elevated blood concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA) and decreased levels of calcium and phosphorous leading to metabolic disorders. Production diseases are the result of cow’s inability to cope with the high metabolic demands of lactation. The term ‘production disease’ encompassed metabolic disorders like ketosis, hypocalcaemia (milk fever) and hypomagnesaemia (grass tetany) but the term has been broadened to include associated clinical diseases like retained placenta, metritis, endometritis, and displaced abomasum (Mulligan and Doherty, 2008). These production diseases are associated with severe economic losses in terms of heavy reduction in milk yield and impaired reproductive performance.
Ketosis is a metabolic problem of dairy animals which often goes undiagnosed. The problem arises due to inability of cattle to fulfil energy demands of production. So animal starts using adipose tissue especially from dorsum of body. This further leads to production of NEFA (Non esterified fatty acid) and ketone bodies (acetone, acetoacetic acid and beta hydroxy butyric acid) in liver (Duffield, 2022). This is compensatory mechanism of body of animal to provide energy. Some of these ketone bodies are used to provide energy but after sometime bad effects start to appear. Ketosis is thought to be more common in high yielding animals between 4 to 6 weeks postpartum. Ketosis is a gateway for LDA, ROP, Mastitis and Metritis.
Symptoms
- Concentrate Refusal 2. Gradual emaciation 3. Decreased Milk Yield 4. Abnormal Licking movements 5. Inappetence
Treatment includes
- Glycerine 2. 50% Dextrose IV 3. Steroids
Diagnosis is very important because sometimes the disease may go unnoticed. So, it is very important to have accurate diagnostic test for ketosis to restore the health and production of animal. Acetone goes off with expiated air so it can’t be measured. Acetoacetic acid can be qualitatively measured in urine and milk. But estimation of BHB (Beta Hydroxy Butyrate) is more accurate method for ketosis diagnosis. So, I used BHB Meter to diagnose ketosis at field level from direct blood. BHB value of more than 1mmol/L is diagnostic for ketosis. I will discuss few cases: 1. Buffalo not taking feed in the evening, decreased milk yield, parturiated 3 months back. BHB 1.8mmol/L, treatment done with dextrose, dexamethasone and glucose precursor. Animal showed recovery after 2 days of treatment. 2. Cow showing gradual emaciation, parturiated 22 days back, decreased feed intake and milk yield (1kg/day). There was history of pneumovagina. BHB 1.6mmol/L. After treatment appetite was restored and milk yield increased to 7kg/day. 3. Cow parturiated 21 days back, showed PPM now showing gradual emaciation and hard faecal balls. BHB 3.4 mmol/L treated with glycerin, steroids and dexamethasone. Animal recovered after 2nd day. 4. Cow calved 1 month back now showing gradual emaciation and decreased milk yield. BHB 1.2mmol/L. The cow showed recovery after 2 days. 5. The cow was showing shivering and abnormal lickings, BHB 2.8mmol/L. Recovery after 3rd day. These cases showed 1. BHB Meter is quite accurate way to diagnose ketosis. 2. Not only high yielders, but even moderate yielders also suffered from ketosis. 3. Ketosis may occur as early as three weeks postpartum.
ROLE OF NEFA AND BHBA IN PREDICTING METABOLIC DISEASES DURING TRANSITION PERIOD
Milk fever (hypocalcaemia)
Incidence rates of clinical hypocalcaemia vary between 3.5 and 7% (De Garis and Lean, 2008). After parturition, there is increased calcium demand for lactation and colostrum production and to meet this demand calcium mobilisation from bones occurs. Milk fever occurs if the calcium homeostasis mechanism which normally maintain blood calcium levels between 9 and 10 mg/dl fails to keep up with the lactational demands resulting in blood calcium levels to fall below 5mg/dl (NRC, 2001). This hypocalcaemia impairs muscle and nerve function to such a degree that the animal is unable to rise. Hypocalcaemia is associated with many other disorders like dystocia, retained placenta, endometritis, infertility, uterine prolapse, mastitis, displaced abomasum, and ketosis (Houe et al. 2001). Intravenous calcium treatments (calcium borogluconate) are used to keep the cow with milk fever alive long enough for calcium homeostatic mechanisms to adapt.
Grass tetany (hypomagnesaemia)
Grass tetany occurs due to low level of magnesium in the blood and is often associated with early lactation as 0.15g magnesium is removed from the blood per litre of milk production. Animals grazing on lush green pastures or potassium fertilized pastures are more prone to grass tetany as potassium interfere with the absorption of magnesium thus leading to deficient levels of magnesium in blood.
Fatty liver and ketosis
During early lactation dietary nutrient supply is unable to meet the nutrient and energy demand of lactation and decreased DMI associated with high estrogen levels and less space in rumen to expand due to foetus leads to negative energy balance (NEB). To cope with the negative energy balance mobilisation of non-esterified fatty acids (NEFA) from adipose tissue occurs. The extent of lipid mobilisation depends on the period of negative energy balance experienced by the animal. Uptake of NEFA by liver is proportional to NEFA concentrations in blood (Bell, 1979). Higher rates of lipid mobilisation led to higher intake of NEFA by liver. Non-esterified fatty acids (NEFA) taken up by liver can either be esterified or oxidised. Esterification of NEFA results in the formation of triglycerides which are either exported as very low-density lipoprotein (VLDL) or stored in liver. The rate at which the export of triglyceride occurs is very slow as compared to other species (Kleppe et al., 1988; Pullen et al., 1990). Thus, leading to accumulation of triglycerides (TG) in liver resulting in fatty liver.
Oxidation of Non-esterified fatty acids (NEFA) lead to either formation of carbon dioxide and ATP or ketone bodies like β-hydroxybutyrate (BHBA). Complete oxidation of NEFA in liver leads to production of energy and carbon dioxide where as incomplete oxidation leads to formation of ketone bodies leading to ketosis. Low levels of insulin enhance fatty acid oxidation by decreasing hepatocyte malonyl CoAwhich in an inhibitor of Carnitine plamitoyltranferase-1 which is responsible for translocation of fatty acid from cytoplasm to mitochondria for oxidation.
Prediction of clinical diseases through NEFA and BHBA levels
Ospina et al. (2010) reported that serum levels of NEFA and BHBA during transition period can be used to predict the occurrence of periparturient diseases like displaced abomasum, ketosis, metritis and retained placenta within 30 days in milk. Ospina et al. (2010) calculated threshold values for both NEFA and BHBA and values higher than the threshold values were associated with the incidence of periparturient diseases or production diseases. Ospina et al. (2010) reported that NEFA concentrations of ≥ 0.29 mEq/L prepartum and ≥ 0.57 mEq/L postpartum were associated with the risk of developing displaced abomasum, metritis, or retained placenta during the first 30d in milk and BHBA concentrations of ≥1.0 mmol/L from day 3 to 14 postpartum were associated with increased risk of clinical ketosis as well as metritis.
Compiled & Shared by- Team, LITD (Livestock Institute of Training & Development)
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