Abstract

Between April 2006 and March 2015, a recent survey in UK commercial dairy herds has shown that approximately half of cows in the last 10 days before calving and ¾ of cows in the first 20 days in milk had evidence of eNEB. However, herd variation is significant, and many herds manage to keep cows with impressive milk yields in satisfactory energy balance around calving. Whatever method of assessment is used for eNEB, it needs to take into account selection of cows for sampling, stage of the production cycle to target “at risk” periods (within 10 days of predicted calving date in dry cows, and the first three weeks of lactation in freshly calved milking cows), group size and use of background information. Interpretation of the results is key, and this requires a holistic view of both the ration and nutritional management.

Introduction

The transition period from 3 weeks pre-calving to 3 weeks after calving is recognised as a critical period during the production cycle of the dairy cow. Not only must the cow calve, but she will also start lactation, which results in a substantial increase in the cow’s energy requirements in a short period of time. It is also the period when the majority of clinical disease issues occur, and it is now recognised that issues with excessive negative energy balance play a significant role underlying many of these transition cow diseases.

Negative Energy Balance (NEB) and excessive Negative Energy Balance (eNEB)

It is considered that almost all cows will undergo a degree of negative energy balance (NEB) in early lactation, as the cow struggles to meet the substantial demands of lactation. This is compounded by a reduction in Dry Matter Intake (DMI) around calving. This NEB will result in a mild mobilisation of body reserves and fat deposition in the liver, but may not be substantial enough to affect cow health and productivity.

However in a proportion of cows, this mobilisation of body reserves progresses to result in excessive negative energy balance (eNEB). Increased levels of β-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFA) in the bloodstream are used as markers of eNEB, where appropriate thresholds are used for interpretation that have been determined in a number of research studies looking at associations with negative effects on cow health, productivity and future fertility. It is worth noting that this situation of eNEB is over and above normal, and such elevated BHB and NEFA levels are associated with harmful effects on cow health and future productivity.

Why does excessive Negative Energy Balance (eNEB) matter?

Recent published results from the Dairy Herd Health and Productivity Service (DHHPS) at the Royal (Dick) School of Veterinary Studies (RDSVS), University of Edinburgh have shown that cows with elevated BHB and NEFA concentrations are common in DHHPS submissions, suggesting that the prevalence of eNEB in UK commercial dairy herds is high. Between April 2006 and March 2015, approximately half of cows in the last 10 days before calving and ¾ of cows in the first 20 days in milk had evidence of eNEB (Figure 1: Macrae et al. 2019). The flip side to this is that ½ of dry cows and ¼ of fresh calvers have good energy results i.e. eNEB is

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not inevitable in modern dairy cows. The variation between herds is significant, and many herds manage to keep cows with impressive milk yields in satisfactory energy balance around calving.

There are now a large number of studies that have associating high (ie. abnormal) BHB and NEFA levels with reduced milk production, increased risk of transition cow diseases (such as LDAs and metritis), and poor reproductive performance (reviewed by Ospina et al., 2013; Raboisson et al., 2014; Benedet et al., 2019)(Figure 2). Whilst clinical ketosis (which can be confirmed by the biochemical detection of BHB levels over 3.0 mmol/l) is comparatively rare and extreme, there are significant harmful effects associated with subclinical ketosis (i.e. raised BHB) and elevated NEFAs on cow health and productivity. For example, one North American study showed that cows with elevated NEFA levels over 0.6 mmol/l in the first 3 - 14 days of lactation had a 647 kg reduction in 305 day milk yield (approximately 2 litres per day during lactation), had a 10 times increased risk of developing an LDA, and 16% reduced likelihood of pregnancy (Ospina et al. 2010a; Ospina et al. 2010b).

Assessment of eNEB

There are a number of different methods that can be used for the assessment of energy balance in cows including:

Body condition score (BCS) and BCS change Liveweight change Analysis of urine and/or milk for BHB concentrations Analysis of blood for various metabolites: primarily BHB, NEFA and glucose, but

cholesterol and IGF-1 have also been identified for the potential assessment of eNEB Analysis of milk for indirect markers of eNEB such as butterfat, milk protein, and

fat:protein ratio Analysis of milk for indirect markers of eNEB using mid-infrared (MIR) spectroscopy Analysis of milk for fatty acid content. Certain long-chain fatty acids will increase due

to increased body fat mobilisation (predominantly C18:0 and C18:1 cis-9), whereas other fatty acids may reduce due to nutrient and energy deficiency.

It is not the purpose of this paper to go through the advantages and disadvantages of all these differing methods. There is much current focus on the development of milk testing through Milk Recording Organisations (MRO), and some of these newer indirect milk assessments are already commercially available for milking cows (but not of course dry cows). This paper will more focus on the rules for assessment of eNEB and importance of correct interpretation, which apply regardless of the method used to assess eNEB.

Studies assessing BHB and NEFA concentrations in blood samples have identified thresholds used for the assessment of eNEB in dairy cows around calving (i.e. levels above which are associated with potentially harmful effects on cow health and productivity). This is not the same as standard laboratory reference ranges that might be encountered in companion animal practice, which are based on ranges where 95% or 97.5% of a clinically healthy population lie. It is important to emphasise this, in that interpretation needs to take into

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account that raised BHB and NEFA values are not “normal”, and thresholds used are associated with potentially negative effects for farm productivity.

However it is also important to recognise that elevated BHB and NEFA concentrations do not inevitably result in harmful effects on the individual cow, as these studies are all based on epidemiologic associations in dairy herds where multiple risk factors are involved. This is best explained by the following fictional example, looking at the association between NEFA levels in late pregnancy and development of LDAs post-partum, illustrated in Table 1.

Group High NEFA group Low NEFA groupNumber of cows 100 cows 100 cowsNumber of LDAs 10 LDAs 1 LDALDA rate 10% 1%

Table 1. Fictional worked example of research studies looking at the association between NEFA levels in late pregnancy and development of LDAs post-partum

As can be seen from this example, the risk of developing an LDA increases significantly for cows with high NEFAs (the Odds Ratio is 11). However, 90% of the cows in the high NEFA group did not develop an LDA, and so having a high NEFA does not guarantee that the cow will develop an LDA.

Previous studies have recommended that NEFAs should only be used for the assessment of eNEB in late pregnancy, and BHB assessment should be used in freshly calved cows (Cook et al. 2006). However this is an over-simplification of the complex metabolic pathways in cattle, and indeed there are studies showing that elevated NEFAs in freshly calved cows are associated with increased risks for the development of periparturient diseases and reduced milk production, compared to high BHBs (Ospina et al. 2013).

In addition, the correlation between BHB and NEFA levels in individual cows is poor in both late pregnancy and lactation (r = 0.119)(see Figure 3), as shown in other studies (Ospina et al. 2013; McCarthy et al. 2015). These metabolites assess different stages of energy metabolism in the cow with NEFAs being a more direct measure of fat mobilisation, whereas BHBs arise when fat is partially oxidised in the liver to form ketone bodies. BHBs can also arise from sources other than fat mobilisation, and as can be seen in Figure 3, it is not uncommon to see cows with high BHBs and low NEFAs, or vice versa.

Although there are some interpretations of cows with Type I (“classic” ketosis in cows 4-6 weeks calved) or Type II ketosis (associated with fatty liver around calving) based on differences in their BHB, glucose and NEFA concentrations (Oetzel 2007), such interpretation is likely to be overly simplistic. Given the complex nature of energy metabolism in the high producing dairy cow, it is probably not surprising that BHB and NEFA levels are poorly associated. Assessment of multiple parameters (BHB, NEFA and glucose) enables better assessment of energy status around calving than reliance on a single measure.

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“Rules” for assessment of eNEB

Regardless of which method is used to assess eNEB (milk or blood analyses, cow-side BHB testing or multiple biochemical measurements), there are some fundamental rules necessary to ensure the validity of any assessment of energy balance in dairy cattle (Whitaker 2004).

1. Timing of testing

Cows should be sampled as far as possible under standard farm conditions, without undue stress or abnormal routine. Cows should not be sampled within 3-4 hours of a large intake of concentrate feed (for example immediately after exiting the parlour if they receive significant amounts of concentrate [over 3 kg per feed] in the parlour), and they should not be left for 3-4 hours without feed prior to sampling (for example if they have been shed off at milking and stood in a pen without food; Figure 4).

Whilst minor changes in diet can be tolerated, it is advisable to leave cows for at least 2 weeks after any major diet change (for example turnout, housing, change in silage clamp or introduction of maize silage) to allow the rumen and cows’ metabolism to settle prior to blood sampling.

Sampling should focus on key “at risk” periods including:

2 weeks after any major changes in diet to check what the cows think of the new diet – e.g. changes in silage clamp, winter housing, decrease in grazing quantity/quality

In block calving herds, sampling is concentrated during the calving period. So a sampling plan for a block calving herd would test at the following timepoints:

o Month prior to calving. Sample “far-off” at 30 days off calving, and “close up” dry cows within 10 days of their predicted calving dates

o First month once calving starts (“close-up” dry cows, fresh calvers in the first 3 weeks of lactation)

o Second month of calving (“close-up” dry cows, fresh calvers, mid lactation cows at 2 months calved)

o If the herd is still calving cows in the third month, then re-sample as above until calving finishes

o Additional sampling can occur if cows are struggling with grazing or mixed diet

If transition cow health issues have been identified, then sampling only groups of “close up” dry cows every 4-6 weeks focuses monitoring on this key period.

2. Stage of production cycle

The two most critical groups to blood sample for the assessment of energy balance in a dairy herd are:

Early lactation group. Most of the research studies assessing cows in early lactation for BHB and NEFA concentrations have sampled cows within the first 2 - 3 weeks of lactation. Some authors do recommend sampling “early lactation” cows later into

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lactation (especially for the assessment of “Type I ketosis”), but the further into lactation the cow goes, the more likely she is to return to positive energy balance as her DMI increases. Therefore sampling early lactation cows too far into lactation runs the risk that BHB and NEFA levels will have returned to below threshold concentrations (as shown in Figure 1), which will result in misclassification of eNEB in these cows.

Can cows be sampled too soon after calving? North American studies have sampled cows either immediately after calving or after 3 days calved for the assessment of eNEB, showing a peak prevalence and incidence at 5 Days in Milk (DIM)(McArt et al. 2012; Ospina et al. 2013). There are concerns that sampling cows in the first 10 days of lactation can lead to difficulties in interpretation when there is a substantial change in diet at calving, and the cows may still be adapting to the milking cow diet and social group. The argument is that leaving cows at least 7 - 10 days into lactation gives the cows time to adjust to the milking cow diet and social group, resulting in more representative biochemical results. However the concern is that by not sampling cows in the first 10 DIM, this may underestimate eNEB prevalence.

“Close up” transition dry cow group. These should be sampled within the last 10 days prior to their predicted calving date, to assess the critical period as DM intakes decline precalving and energy requirements increase due to colostrogenesis, putting the precalving cow at an increased risk of eNEB. Cows due to calve imminently or carrying twins should not be sampled if at all possible.

Whilst these are the two most critical groups, a mid lactation group at 90 – 120 days calved can also be valuable as a “control group” in the milking cows. These cows will be in the same feeding group as the early lactation cows, and will be past peak lactation with good milk yields and DMI. They therefore serve as a good control group to assess the base milking cow diet in cows with good DMI, and help to differentiate between Type I and Type II ketosis issues.

3. Group size

Sampling for the assessment of eNEB cannot be an individual cow test, due to individual cow variation in milk yield, metabolism and feed intakes. Sampling individual cows for eNEB is only useful when screening freshly calved cows for clinical disease (such as ketosis) and directing appropriate treatment.

The number of cows that require to be sampled in the group varies depending on the group size of the population “at risk”, estimated prevalence of eNEB (as assessed using BHB and NEFA), and the required confidence in the result. North American guidelines for the assessment of energy balance in large herds at 95% confidence intervals recommend sampling a minimum of 12 – 20 cows in the “at risk” groups (ie. within 10 days of calving, and early lactation 3 – 14 Days in Milk)(Oetzel 2007; Ospina et al. 2013). However in smaller UK dairy herds calving all year round, it can be difficult to have sufficient cows available for sampling in the “at risk” group. In such circumstances with smaller “at risk” group sizes of 10

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cows, a minimum group size of 5 – 8 cows would be considered sufficient to detect 20% prevalence of eNEB at a much lower 75% confidence interval (LeBlanc 2010). However practitioners must be aware that reducing the sample size will increase the inaccuracy and potential misclassification of herds with eNEB. For example, if a group of 3 cows have been sampled and all have normal metabolites, there is still the possibility (indeed probability) of missing significant issues with eNEB.

4. Selection of cows for testing

It is important that cows selected for blood sampling are representative of the group and herd. Cows that have clinical disease problems such as metritis or lameness are likely to have evidence of eNEB secondary to their disease problems, and should not be sampled.

It should also be noted that level of milk production can also affect the prevalence of eNEB observed. For example first lactation heifers are significantly less likely to have elevated BHB and NEFA results due to their lower levels of milk production, whereas older higher yielding cows suffer from a higher prevalence of eNEB (Macrae et al. 2019b). Sampling poorly performing low yielding cows may be informative if they are not milking to expectation due to issues with eNEB or metabolic disease. However cows with poor production due to other reasons (for example lack of dietary protein) can have good biochemical energy parameters as their lower levels of milk production result in lower energy requirements, which are readily met from the diet.

5. Use of background information

As will be discussed later in the interpretation section, the diagnosis of a herd having problems with eNEB is only the start. Interpretation of the results requires additional information on the cows sampled (including stage of lactation, milk yield, bodyweight, body condition score, lactation number), ration fed and nutritional management. Such background information is critical to the correct diagnosis of any problems, and implementation of changes to sort any problems identified.

Interpretation

The accurate diagnosis of a herd having problems with eNEB (by whatever method) is only the beginning of the process. What we are really interested in is identifying is why and where the problem has arisen from, and what the most cost-effective solution is. To illustrate this, there are a number of different reasons why cows in early lactation might have evidence of eNEB, including:

Inadequate energy content of the diet. This situation can be commonly encountered on farms with high genetic merit for production cows at grass, where grazing quality and/quantity is not sufficient to support high milk yields (Figure 5). Poor quality conserved forages may also present problems, or levels of concentrate feeding may be reduced for apparent economic reasons.

Inadequate food intakes (of a diet that does contain sufficient energy density). DMI may be restricted due to poor palatability of the diet (if it is heating or spoiling for

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example), or restricted feed access due to insufficient trough space and overcrowding (Figure 6). High levels of lameness may also restrict feed intakes. DMI are critical in high yielding dairy cows for them to be able to eat enough diet to meet the high demands of lactation. In high yielding herds, small intake restrictions may have significant consequences for DMI in freshly calved cows, who already have compromised feed intakes and are lower down the herd social order.

Poor digestion and utilisation of available energy from the diet. If there is too much fermentable carbohydrate (i.e. starch and sugar) and too little physically effective fibre in the diet, then rumen health becomes compromised. This results in poor digestion of the diet, and undigested food material passes through the gut (and often observed in the cows’ faeces). As the cow cannot get sufficient energy to meet the demands of milk production, affected cows show evidence of eNEB.

Poor transition dry cow management in late pregnancy. If there are substantial problems with late pregnancy nutrition (for example excessive BCS, under or over-feeding of energy, restricted intakes), then this will carry over to affect energy balance in early lactation.

As can be seen, these are four very different reasons for raised BHB and NEFA values (i.e. eNEB) in freshly calved cows, with four very different solutions. For example, the solution for “inadequate energy content of the diet” is to feed more concentrates. However doing this for “poor digestion and utilisation of available energy from the diet” could make the situation worse. This is where interpretation with the available farm information is key, to ensure that the correct interpretation is made, and the right solution is put in place. The practicing veterinary surgeon with a holistic view of the herd should be in the best position to co-ordinate advice on this, in conjunction with the farm staff and nutritional advisor.

Conclusions

Excessive negative energy balance is common in UK dairy herds, and is associated with substantial harmful effects on productivity, cow health and future fertility. Assessment of energy balance can be achieved using a number of methods, but it is important to sample representative groups of cows during the key “at risk” periods of late pregnancy and early lactation. Correct interpretation of any assessment requires the use of background information on the cows and their nutritional management, in order that appropriate corrective actions can be undertaken.

Keywords

dairy cow, assessment, negative energy balance, betahydroxybutyrate, non-esterified fatty acid, glucose

Key points

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Excessive negative energy balance affects around ½ of UK dairy cows in late pregnancy, and ¾ of cows in the first 20 days after calving

Such excessive negative energy balance is associated with harmful effects on cow health, milk production and fertility

Groups to sample for the assessment of energy balance should include: 1) dry cows within the last 10 days of pregnancy 2) freshly calved cows in the first 3 weeks of lactation and 3) a control group of mid lactation cows at 90 – 120 days calved

Cows for sampling should be representative of the group and herd It is essential to use background information on the cows and their nutritional

management to correctly diagnose problems and implement corrective control measures

References

Benedet A, Manuelian CL, Zidi A, Penasa M, De Marchi M. 2019. Invited review: beta-hydroxybutyrate concentration in blood and milk and its associations with cow performance. Animal. 13(8):1676–1689. doi:10.1017/S175173111900034X.

Cook N, Oetzel G, Nordlund K. 2006. Modern techniques for monitoring high producing ‐dairy cows 2. Practical applications. Pract . 28(10):598–603. doi:10.1136/inpract.28.10.598.

LeBlanc S. 2010. Monitoring metabolic health of dairy cattle in the transition period. J Reprod Dev. 56 Suppl:S29-35. doi:10.1262/jrd.1056S29.

Macrae AI, Burrough E, Forrest J, Corbishley A, Russell G, Shaw DJ. 2019a. Prevalence of excessive negative energy balance in commercial United Kingdom dairy herds. Vet J. 248:51–57. doi:10.1016/j.tvjl.2019.04.001.

Macrae AI, Burrough E, Forrest J, Corbishley A, Russell G, Shaw DJ. 2019b. Risk factors associated with excessive negative energy balance in commercial United Kingdom dairy herds. 250:15–23. doi:10.1016/j.tvjl.2019.06.001.

McArt JAA, Nydam DV, Oetzel GR. 2012. Epidemiology of subclinical ketosis in early lactation dairy cattle. J Dairy Sci. 95(9):5056–5066. doi:10.3168/jds.2012-5443.

McCarthy MM, Mann S, Nydam DV, Overton TR, McArt JAA. 2015. Short communication: Concentrations of nonesterified fatty acids and β-hydroxybutyrate in dairy cows are not well correlated during the transition period. J Dairy Sci. 98(9):6284–6290. doi:10.3168/jds.2015-9446.

Oetzel GR. 2007. Herd-Level Ketosis – Diagnosis and Risk Factors. In: American Association of Bovine Practitioners. Preconference Seminar 7C: Dairy Herd Problem Investigation Strategies: Transition Cow Troubleshooting. Vancouver, BC. p. 67–91.

Ospina PA, McArt JA, Overton TR, Stokol T, Nydam D V. 2013. Using nonesterified fatty acids and betahydroxybutyrate concentrations during the transition period for herd-level monitoring of increased risk of disease and decreased reproductive and milking performance. Vet Clin North Am - Food Anim Pract. 29(2):387–412. doi:10.1016/j.cvfa.2013.04.003.

Ospina PA, Nydam DV, Stokol T, Overton TR. 2010a. Associations of elevated nonesterified

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fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States. J Dairy Sci. 93(4):1596–1603. doi:10.3168/jds.2009-2852.

Ospina PA, Nydam DV, Stokol T, Overton TR. 2010b. Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases. J Dairy Sci. 93(2):546–554. doi:10.3168/jds.2009-2277.

Raboisson D, Mounie M, Maigne E. 2014. Diseases, reproductive performance, and changes in milk production associated with subclinical ketosis in dairy cows: a meta-analysis and review. J Dairy Sci. 97(12):7547–7563. doi:10.3168/jds.2014-8237.

Whitaker, D.A., 2004. Metabolic Profiles, in: Andrews, A.H., Blowey, R.W., Boyd, H., Eddy, R.G. (Eds.), Bovine Medicine: Diseases and Husbandry of Cattle. Blackwell Science, Oxford, pp. 804–817.

Further information available at www.ed.ac.uk/vet/dhhps

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Figure 1. Prevalence of eNEB in commercial UK dairy herd dataset restricted to -50 days relative to calving to 200 DIM for (a) β-hydroxybutyrate (BHB); (b) non-esterified fatty acids (NEFA); (c) Glucose; (d) NEFA and/or BHB; (e) NEFA and/or BHB and/or glucose. Each bar represents a 10-day block. Vertical error bars represent 95% confidence interval (CI), and the best fit as a thick black line. Figure adapted with permission from Macrae et al., 2019.

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Figure 2. Diseases such as metritis and subsequent endometritis may be one sign of underlying issues with eNEB around calving.

Figure 3. Poor correlation between BHB and NEFA levels in individual cows during lactation and dry period (same dataset as Macrae et al., 2019a; r = 0.119).

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Figure 4. Restricting cows without food for 3-4 hours prior to sampling can result in abnormal biochemical values and subsequent misinterpretation of results.

Figure 5. A combination of poor quality grazing and inappropriate buffer feeding will result in eNEB

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Figure 6. Poor feed bunk management and overstocking will restricting feed intakes in freshly calved cows, resulting in eNEB

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CPD MCQs

1. According to published UK results in 2019, approximately how many dry cows show evidence of excessive negative energy balance (eNEB) in the last 10 days before calving?

a. 0%b. 25%c. 50%d. 75%e. 100%

Correct answer: c

2. According to published UK results in 2019, approximately how many cows have good energy results (ie. normal BHB, glucose and NEFA concentrations) in the first 20 days of lactation?

a. 0%b. 25%c. 50%d. 75%e. 100%

Correct answer: b

3. You wish to sample a herd for the assessment of energy balance. However this year’s maize silage has just been introduced into the milking cow diet at a high inclusion level. How long should you leave the herd to settle on the new diet before sampling?

a. 1 dayb. 1 weekc. 2 weeksd. 3 weekse. 1 month

Correct answer: c

4. You have arranged to blood sample a herd for the assessment of energy balance. However you get called to a calving first, and so are late to arrive on the farm. The cows have been shed off into a yard with no food after milking for blood sampling. What is the approximately length of time that cows can be without feed prior to blood sampling before it starts to affect their biochemical results?

a. Under 1 hourb. 1-2 hoursc. 2-3 hoursd. 3-4 hourse. 4-5 hours

Correct answer: d

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5. What are the recommendations for days relative to predicted calving date for sampling dry cows in late pregnancy for the assessment of excessive negative energy balance?

a. Within 10 days of predicted calvingb. 10 – 20 days off predicted calvingc. 20 – 30 days off predicted calvingd. 30 – 40 days off predicted calvinge. 40 – 50 days off predicted calving

Correct answer: a

6. You have arranged to sample a dairy herd for the assessment of energy balance. Which freshly calved cow should the farmer present for blood sampling?

a. Cow 41, calved 37 days ago with no reported health issuesb. Cow 87, calved 15 days ago, giving 5 litres of milkc. Cow 183, calved 10 days ago, operated on to correct an LDA 3 days agod. Cow 297, calved yesterday with twinse. Cow 924, calved 14 days ago with no reported health issues

Correct answer: e

7. When sampling cows for the assessment of energy balance, what additional background information is needed for the correct interpretation of the results?

a. Daily milk yieldb. Foot trimming recordsc. Individual cow Johne’s Disease serology resultsd. Individual Cow Somatic Cell Counte. Previous fertility treatments

Correct answer: a

8. Which lactation group have a significantly lower prevalence of excessive negative energy balance?

a. First lactationb. Second lactationc. Third lactationd. Fourth lactatione. Fifth lactation

Correct answer: a

9. How significant is the correlation between BHB and NEFA levels in individual cows for the assessment of excessive negative energy balance?

a. Good in both milking and dry cowsb. Good in milking cows, poor in dry cowsc. Poor in milking cows, good in dry cowsd. Poor in both milking and dry cowse. NEFAs should not be assessed in milking cows

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Correct answer: d

10. Cow 287 has been blood sampled at 35 days calved for the assessment of energy balance, giving 42 litres of milk, body condition score 1.5 out of 5. Her BHB, glucose and NEFA concentrations are within the optimum thresholds used. What is the most likely explanation of the interpretation of the biochemical results in this cow?

a. Her biochemical results are as expected in such a high yielding cowb. She has always been in satisfactory energy balance since calvingc. She had excessive negative energy balance previously in the first 3 weeks of

lactationd. She is showing current evidence of excessive negative energy balancee. There is an error with the biochemical testing for BHB, glucose and NEFA

Correct answer: c

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