Comp Clin Pathol

DOI 10.1007/s00580-016-2377-z

ORIGINAL ARTICLE

The effect of subclinical ketosis on indices of insulin sensitivity

and selected metabolic variables in transition dairy cattle
Mohamed A. Youssef 1 & Maged R. El-Ashker 1 & Marwa S. Younis 2

Received: 15 August 2016 / Accepted: 16 November 2016

# Springer-Verlag London 2016

Abstract Up to now, there have been several endeavors de- (P = 0.007). The elevated values of serum glucose, insulin,
scribing the extent of insulin resistance (IR) in dairy cows with cortisol, NEFA, and BHBA are therefore suggestive of im-
particular emphasis on transition period, but it is not fully paired whole-body insulin sensitivity and were consistent with
explicit what IR plays in the etiology of subclinical ketosis decreased RQUICKI in the diseased cows. Further studies are
(SCK) in transition cows. The present study was therefore needed to use prophylactic feed additives to enhance insulin
conducted on a commercial dairy herd with a stock population sensitivity and to help mitigate the deleterious consequences
of 730 Holstein-Friesian cows to evaluate the extent of IR in of bovine ketosis during the transition phase.
transition cows with SCK. Blood samples were collected ini-
tially from each of the transition cows (n = 24) to estimate Keywords Subclinical ketosis . Insulin resistance . Dairy
serum levels of β-hydroxybutyrate (BHBA). Cows with no cattle . Transition period
clinical signs but having serum BHBA >1.20 to 2.9 mmol/l
were considered to have SCK (n = 20), while those having
serum BHBA concentrations <1.00 mmol/l were considered Introduction
as controls (n = 4). Blood samples were then used for estimat-
ing values of serum non-esterified fatty acid (NEFA), glucose, The periparturient period, defined as 3 weeks around the time of
insulin, and cortisol. Different surrogate indices for insulin parturition, is physiologically stressful and represents a critical
sensitivity were also calculated. Our findings demonstrated time for dairy cattle (Sundrum 2015). During that time, many
that values of BHBA, NEFA, and insulin were significantly physiological, nutritional, metabolic, and immunological alter-
higher in cows with SCK than those of controls (P < 0.05), ations can occur as the cow transits from a gestational non-
while values of cortisol and glucose, despite being higher in lactating state to the period of copious milk production
diseased cows than controls, did not reach statistical (Sordillo and Raphael 2013). The ability of a dairy cow to
significance (P = 0.605 and 0.269). Indices of insulin sensi- withstand these alterations is crucial to optimize herd manage-
tivity showed a significant decrease in revised quantitative ment since the demands for milk production cannot be met
insulin sensitivity check index (RQUICKI) (P = 0.047) solely by feed intake (Herdt 2000). Cattle unable to safely tran-
and a significant increase in homeostasis model assessment sit through this period are at a higher risk for metabolic disor-
(HOMA) in diseased cows compared with controls ders and decreased milk yield (Drackley 1999; Herdt 2000).
Among the commonly reported metabolic disorders that
occurred during the transition phase, hyperketonemia is most-
* Maged R. El-Ashker ly prevalent and provokes a significant economic impact
[email protected] (Opsomer 2015). In that context, a working definition of
SCK is that Ba condition indicated by abnormal concentrations
1
Department of Internal Medicine and Infectious Diseases, Faculty of of circulating ketone bodies in the absence of detectable clin-
Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt ical signs of the disease^ (Andersson 1988). In a dairy cattle
2
Animal Health Research Institute-Mansoura Provincial Laboratory, herd, SCK can lead to serious economic loss involving a de-
Mansoura, Egypt crease in milk yield, decrease reproductive efficiency, and
 Comp Clin Pathol

increase the risk of various diseases such as abomasal dis- farm had, in general, an average of 305 days normalized
placement and clinical ketosis (Andersson 1988; Opsomer milk production, body weight of 550 ± 50 kg, and at 3–
2015). Hence, early diagnosis of SCK can permit proper treat- 7 years of age and where economics played the main role in
ment and help mitigate further economic losses (Enjalbert farm decisions. The animals were apparent healthy, of
et al. 2001; Geishauser et al. 2001). Because of its stability different parities, and were artificially inseminated. The
in blood, beta-hydroxybutyrate (BHBA) still considered the cows milked twice a day with a record of milk production in
gold standard test for the diagnosis of SCK under field condi- an average of 20.0 ± 3.0 kg/head/day in summer season and
tion (Herdt 2000). had a range of body condition score at 3–3.5. The investigated
Insulin resistance (IR) has been defined as a state when a cows were free from external and internal parasites and had no
physiological level of insulin produces a lesser biological re- history of metabolic and production disease in that year. All
sponse than do normal (Kahn 1978). Other investigators have animals were kept under identical conditions of housing and
described IR as a condition in which high amount of insulin is vaccination throughout the study period. The feed for all cows
required to produce a normal biological response (Berson and consisted of a base ration fed as a daily total mixed ration as
Yalow 1970). It has been reported that insulin could play a well as corn silage and Alfa hay in summer as an alternative
fundamental role in the physiological adaptation of dairy cows source of roughage. Water supply was offered to all animals
especially around the time of parturition (Zachut et al. 2013). ad libitum. Cows were dried off 60 days before the expected
The glucose metabolism in ruminants is often characterized by time of parturition.
low peripheral glucose levels and a low insulin response of the
peripheral tissues (Hayirli 2006). After calving, cows undergo Sampling and the criteria of animal selection
a period of transient IR to prioritize the insulin-independent
uptake of glucose by the mammary gland in order to favor On the farm, there were 170 cows at different stages of lacta-
milk production. Hence, the maintenance of blood glucose tion. Twenty-four primiparous cows (i.e., cows pregnant with
values within the normal physiologic ranges is of utmost im- their second calf), at 3–4 years of age, were only located at a
portance during the transition phase (De Koster and Opsomer late gestation time approximately 3 weeks before the expected
2013). time of parturition. For the initial screening of SCK, 10 ml of
Factors that cause IR in humans are also related to those venous blood were drained from each of the transition cattle
involved in the development of ruminant hepatic lipidosis and (n = 24), through coccygeal venipuncture into a plain tube,
ketosis (Drackley et al. 1992). The mechanisms for IR are not i.e., without anticoagulants. Blood was rapidly cooled on
completely understood and seem to be a combination of ge- crushed ice and was transported to the laboratory to be centri-
netic, cellular, and environmental causes (Ferrannini and Mari fuged at 1400×g for 10 min to separate blood serum. Only
1998). To date, there have been several endeavors describing clear non-hemolyzed serum was collected and then aliquoted
IR in dairy cows with emphasis on transition or lactation pe- for estimation of BHBA. Basically, cows with no remarkable
riods (Sano et al. 1993; Sternbauer and Luthman 2002; Terao clinical signs but having serum BHBA concentrations >1.20
et al. 2010; Jaakson et al. 2013) or in association with primary to 2.9 mmol/l were considered to have SCK as previously
clinical ketosis in cows (Xu et al. 2014) or ketotic cows with mentioned by McArt et al. (2012) and Xu et al. (2014). At
primary displaced abomasum (Khalphallah et al. 2015). But, it the meantime, cows with no clinical signs and serum BHBA
is not fully explicit what IR plays in the etiology of SCK. In concentration <1.00 mmol/l were considered to be negative
line with these considerations, the present study was designed controls. The collected serum samples were further used to
to evaluate the extent of IR in cows with SCK during the estimate the following biochemical variables: non-esterified
transition period. fatty acid (NEFA), glucose, insulin, and cortisol, according
to the standard protocols of the manufacturers. Commercial
kits were used for estimating serum BHBA (Ben Biochemical
Materials and methods Enterprise, Italy; Cat No. HB8855), glucose (Spinreact, Spain;
Cat No. 41011), insulin (Bovine Insulin ELISA Kits, ALPCO,
Study area and animal population USA; Cat No. 30-AS1011.1), and cortisol (Bovine cortisol
ELISA Kit, MyBioSource, USA Cat No. MBS738051), while
The present study was conducted on a commercial herd with a NEFA was measured chemically according to the method de-
stock population of 730 Holstein-Friesian cattle. The farm is scribed by Schuster and Pilz (1979). In general, cows were
located in the city of Ras El-Bar, Damietta Governorate, included in the present study in the following conditions: (1) if
Egypt. The study has taken place during the period between their previous gestation period was more than 260 days, (2) if
August and October 2015 where the average annual tempera- they were not treated for ketosis before BHBA testing, and (3)
ture was 20.2 °C (26 °C in August and 23 °C in October) if they have had a previous normal calving altitude and were
(http://en.climate-data.org/location/51100/). Cows on the being clinically healthy following the parturition.
Comp Clin Pathol

Measurements of insulin sensitivity Table 2 Means ± SD of insulin sensitivity indices in dairy cattle with
subclinical ketosis compared with controls during the transition period

Different surrogate indices were used to calculate insulin sen- Index Control group (n = 4) SCK group (n = 20) P value
sitivity as follows: revised quantitative insulin sensitivity
check index (RQUICKI) = 1 / [log (glucose, mg/dl) + log RQUICKI 0.35 ± 0.013 0.32 ± 0.027 0.047
(insulin, μU/ml) + log (NEFA, mmol/l)] (Xu et al. 2014); QUICKI 0.30 ± 0.009 0.29 ± 0.02 0.354
quantitative insulin sensitivity check index (QUICKI) = 1 / RQUICKIBHB 0.34 ± 0.022 0.55 ± 0.36 0.224
log (glucose, mg/dl) + log (insulin, μU/ml) (Katz et al. HOMA 100.8 ± 19.9 161.8 ± 79.02 0.007
2000); revised quantitative insulin sensitivity check index in- Log HOMA 1.95 ± 0.13 2.13 ± 0.26 0.200
cluding BHBA (RQUICKIBHB) = 1 / log (glucose, mg/dl) + HOMA−1 0.0098 ± 0.002 0.0083 ± 0.006 0.630
log (insulin, μU/ml) + log (NEFA, mmol/l) + log (BHBA,
RQUICKI revised quantitative insulin sensitivity check index, QUICKI
mmol/l) (Balogh et al. 2008; Abuelo et al. 2016); homeostasis quantitative insulin sensitivity check index, RQUICKIBHB revised quan-
model assessment (HOMA) = insulin (μU/ml) × glucose titative insulin sensitivity check index including BHBA, HOMA homeo-
(mmol/l) (Abuelo et al. 2016); log transformation of HOMA stasis model assessment, Log HOMA log transformation of HOMA,
(log HOMA) = log (insulin (μU/ml) × glucose (mmol/l) HOMA−1 reciprocal score of HOMA, SCK subclinically ketotic group
(Abuelo et al. 2016); and reciprocal score of HOMA
(HOMA −1 ) = 1 / (insulin (μU/ml) × glucose (mmol/l) BHB < 1.00 mmol/l were served as controls (n = 4). Cows
(Abuelo et al. 2016). with SCK had no detectable clinical findings during the initial
screening. Biochemically, values of serum BHBA, NEFA, and
insulin were significantly higher in cows with SCK than those
Statistical analysis of controls (P < 0.05), while values of serum cortisol and
glucose (despite being higher in diseased cows than those of
Data were statistically analyzed by using SPSS statistical soft- controls) showed no significant changes between the two
ware program (SPSS, version 15, USA). Independent samples groups (P = 0.605 and P = 0.269, respectively) (Table 1).
t test was used to compare variables between ketotic and con- Indices of insulin sensitivity showed a significant decrease
trol groups. The means and standard deviation (SD) for each in RQUICKI (P < 0.047) and a significant increase in HOMA
variable were calculated. At P < 0.05, results were considered (P < 0.007) in diseased cows compared with controls, while
statistically significant. Spearman correlation coefficient was other surrogate indices showed no significant alteration be-
also applied to emphasize the correlation among the tested tween the two groups (Table 2). A positive correlation be-
variables and the indices of insulin sensitivity. tween BHBA and NEFA (r = 0.44, P < 0.05) was illustrated
in Table 3. There were also statistically significant correlations
among the estimated metabolic variables and indices of insu-
Results lin sensitivity; the detailed results about these correlations are
presented in Table 3.
An overview of biochemical alterations as well as surrogate
indices of insulin sensitivity in dairy cattle with SCK during
the transition period is presented in Tables 1 and 2. In the Discussion
present study, SCK was diagnosed on the basis of serum levels
of BHBA. Cows with serum BHBA >1.20 mmol/l were con- In the present study, cows with SCK showed a statistically
sidered to have SCK (n = 20), while cows whose serum significant higher values of serum insulin, BHBA, and
NEFA than those of controls, while values of serum glucose
Table 1 Means ± SD of selected metabolic profile in dairy cattle with and cortisol showed no significant alterations between the two
subclinical ketosis compared with controls during the transition period
groups. Unlike the results obtained in the present study, some
Variables Control group SCK group P value authors have found hypoinsulinemia and hypoglycaemia in
(n = 4) (n = 20) several clinical entities including SCK in cows (Tehrani-
Sharif et al. 2012), clinical ketosis in cows (Kerestes et al.
BHBA (mmol/l) 0.97 ± 0.112 1.8 ± 0.356 0.000
2009; Xu et al. 2014), clinical ketosis in buffalo (Teli and
NEFA (mmol/l) 0.34 ± 0.04 0.50 ± 0.10 0.006
Ali 2007), or physiologically during the transition phase
Glucose (mg/dl) 44.62 ± 4.85 53.16 ± 15.46 0.269
(Abuelo et al. 2016). Nevertheless, some authors have found
Insulin (μU/ml) 40.85 ± 4.94 53.72 ± 18.27 0.014
normal glucose concentrations in cows with SCK (Grohn et al.
Cortisol (ng/ml) 12.58 ± 1.87 14.14 ± 5.79 0.605
1983; Sakha et al. 2006, 2007; Forslund et al. 2010). The
BHBA β-hydroxy butyrate, NEFA non-esterified fatty acid, SCK subclin- enormous glucose drain toward the udder and the distinctive
ically ketotic group features of the transition period in dairy cows make the
 Comp Clin Pathol

Table 3 The correlations among the metabolic variables and indices of insulin sensitivity in dairy cattle with subclinical ketosis during the transition
period

BHBA NEFA Glucose Insulin Cortisol RQUICKI QUICK RQUIKIBHB HOMA Log HOMA HOMA−1

BHBA 1 0.44* −0.06 0.03 0.07 −0.06 0.24 −0.34 0.01 −0.09 0.09
NEFA 1 −0.22 0.06 −0.06 −0.22 0.26 −0.33 −0.05 −.20 0.49*
Glucose 1 0.25 0.31 −0.55** −0.62** −0.22 0.78** 0.71** −0.47*
Insulin 1 0.30 −0.85** −0.75** 0.12 0.78** 0.82** −0.53**
Cortisol 1 −0.27 −0.22 −0.47* 0.38 0.26 0.03
RQUICKI 1 0.82** −0.01 −0.89** −0.89** −0.49*
QUICKI 1 −0.22 −0.81* −0.93** 0.69**
RQUICKIBHB 1 −0.06 0.12 −0.41*
HOMA 1 0.94** −0.61
Log HOMA 1 −0.71**
HOMA−1 1

BHBA β-hydroxy butyrate, NEFA non-esterified fatty acid, RQUICKI revised quantitative insulin sensitivity check index, QUICKI quantitative insulin
sensitivity check index, RQUICKIBHB revised quantitative insulin sensitivity check index including BHBA, HOMA homeostasis model assessment, Log
HOMA log transformation of HOMA, HOMA−1 reciprocal score of HOMA
*Correlation is significant at 0.05 level, **correlation is significant at the 0.01 level

glucose metabolism an example of how intensive genetic se- to the difficulties in synthesizing cortisol when their energy
lection can drive metabolism to extremes. However, it be- demands increase in peak lactation.
comes apparent that glucose could have a low sensitivity The sensitivity of the peripheral tissue to insulin has been
and cannot be a good criterion for the diagnosis of SCK, and assessed through the calculation of various surrogate indices
it does not appear to be useful for monitoring SCK. Similar according to recently published report (Abuelo et al. 2016).
findings were previously obtained by Sakha et al. (2006). It The widely accepted gold standard method for assessing pe-
has also been stated that the periparturient period in dairy ripheral insulin sensitivity is the use of hyperinsulinemic
cows could be associated with alterations in insulin action euglycemic clamp test, but it has been found to be time-
particularly in the peripheral tissues and the extent of insulin consuming and being an invasive procedure; hence, it has
secretion prepartum has been found to correlate with higher not been recommended for use either under field conditions
insulin action (Zachut et al. 2013). or on a large-scale epidemiological investigations (Muniyappa
In the current investigation, BHBA was found to correlate et al. 2008).
significantly with NEFA (r = 0.44, P < 0.05). However, the In the current investigation, cows with SCK showed a sig-
negative correlation between BHBA and glucose concentra- nificant low RQUICKI index and high HOMA compared with
tions was not observed here. These findings were coincided controls. High HOMA level could indicate an increase in se-
with those previously mentioned by Asl et al. (2011), but rum values of glucose, insulin, or both, thereby could suggest
unlike those obtained by several authors elsewhere (Sakha a low insulin sensitivity and low glucose tolerance, while low
et al. 2007; Tehrani-Sharif et al. 2012). In some reports, it RQUICKI and RQUICKIBHB could reflect the high concen-
has been suggested that plasma BHBA concentrations above tration of insulin, glucose, BHBA, and NEFA and hence could
1.4 mmol/l combined with plasma glucose values of less than suggest a low insulin responsiveness. The obtained findings
55 mg/dl could be used to classify cows as having poor energy were in parallel with the recently published reports in
status (Whitaker et al. 1993). periparturient dairy cows (Abuelo et al. 2016). Similarly, some
Our findings showed that serum cortisol despite being authors have found that values of RQUICKI were lower in
higher in cows with SCK than those of controls, its values ketotic cows than those in controls (Xu et al. 2014). Likewise,
did not reach statistical significance (P = 0.605). indices of log HOMA, HOMA−1, and QUICKI were not sig-
Nonetheless, serum cortisol was found to correlate positively nificantly changed between the diseased and control group.
with NEFA (r = 0.63; P 0.01) and glucose (r = 0.42; P 0.05). These findings were a little bit away from the results obtained
The insignificant elevation of circulating cortisol besides the by several other authors (Abuelo et al. 2014, 2016), probably
decrease in the sensitivity of peripheral tissue to insulin (as due to the low number of animals being studied.
demonstrated in this study) could result in a state of hypergly- Several other researchers have recently highlighted the role
cemia. Nevertheless, the obtained results were away from that of NEFAs in insulin sensitivity of dairy cattle. In that context,
obtained by Forslund et al. (2010) who stated that cows with an increase of circulating NEFAs has been detected following a
ketosis had significantly low levels of cortisol attributed that fasting period or by the intravenous administration of a tallow
Comp Clin Pathol

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