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Energy and Mineral Metabolism, Oxidative Stress, Haemogram and Biochemical Profile in Crossbred Cows Suffering From Subclinical Ketosis

Gagandeep Singh S. N. S Randhawa N. Chand S. K. Uppal C. S. Randhawa
Vol 8(2), 275-284
DOI- http://dx.doi.org/10.5455/ijlr.20170810060239

A total of 22 subclinical ketotic and 18 healthy cows were studied to evaluate changes in various metabolic parameters. Blood samples were collected thrice from each cow. In comparison to control cows a significant decrease was seen in haemoglobin, packed cell volume, total erythrocyte count and glucose, (lower than the normal range), and significant increase in beta hydroxyl butyric acid (BHBA) (>1.2 mmol/l) and non esterefied fatty acid (NEFA) (>0.4 mmol/l) indicating subclinical ketosis, where as non significant difference was recorded in all other parameters in sub clinical ketotic cows. Oxidative stress parameters evaluation revealed a significant increase in LPO levels, while a significant decrease was recorded in SOD and GSH levels in subclinical ketotic cows in comparison to the control cows. A significant decrease in body condition score and in milk yield was recorded in SCK cows in comparison to control cows.


Keywords : Body Condition Scoring BHBA GSH Hematolobiochemical LPO Minerals NEFA SOD

Introduction

During the periparturient period, dairy cows undergoes remarkable changes in their metabolism from late pregnancy up to early lactation period, which is characterized by marked changes in their endocrine status and a reduction in feed intake when nutrient demand for the developing conceptus and for the initiation of milk production is increasing (Grummer, 1995). Thus the most of the high producing dairy cows faces negative energy balance (NEB) during this period and the extent of negative energy balance is related to the incidence and severity of various metabolic disorders, such as ketosis, fatty liver, displaced abomasum,  retained placenta, and susceptibility to infections (Grummer et al., 1994). It has also seen that the sub clinical form of these diseases are more dangerous than the clinical form of disease, both at the individual animal level as well as at the herd level, because with the subclinical disease the animals continues to produce at a reduced rate without showing any clinical sign resulting into the significant economic losses to the farmers both in the form of reduced production and with the continuous health loss of the animal. As very less work has been carried out on subclinical ketosis and its effect on various metabolic parameters, therefore the work was planned to study the alterations is the metabolic parameters in cows suffering from subclinical ketosis as well as to study the production losses in these cows.

Materials and Methods

A total of 22 cows suffering from subclinical ketosis, during the early lactation period, without having any clinical signs and 18 healthy cows were evaluated to study the alterations in various metabolic parameters. Of the 22 cows affected with SCK, 20 cows were in 2nd -5th parity and 2 cows were having >5th parity. Blood samples were taken thrice from each cow, (i) Far off dry (FOD)- >10 days following dry off and not < 30 days prior to calving, (ii) close up dry (CUD)- between 3 and 21 days prior to calving, (iii) fresh – 3 to 30 days in milk to evaluate the changes in the values of various  parameters of CMPT  viz. haemoglobin (Hb), packed cell volume (PCV), total erythrocyte count (TEC), total leucocyte count (TLC), total plasma protein (TPP), albumin, plasma urea nitrogen (PUN), creatinine, glucose, Beta- hydroxyl butyric acid (BHBA), non esterified fatty acid (NEFA), calcium (Ca), magnesium (Mg), plasma  inorganic phosphorus (Pi),  sodium (Na), potassium (K), copper (Cu), iron (Fe) and zinc (Zn).  Experiment was conducted with the permission of the institutional animal ethical committee. All the haematological parameters (Hb, PCV, TEC and TLC) were estimated in whole blood on Hematology Analyzer, USA.

Various biochemical parameters (TPP, albumin, PUN, creatinine and glucose) were accessed on semi automatic biochemistry analyzer using kits both plasma BHBA and NEFA levels were estimated on plasma samples in the ELISA plates with the help of kits. The plasma samples(Ca, Mg, Na, K, Cu, Fe and Zn) were digested as per the procedure described by Kolmer et al. (1951) and the digested samples were estimated by Atomic Absorption Spectrophotometer (AAS), where as plasma Pi was determined by using method given by Tausky and Shorr (1953) and the readings were taken using spectrophotometer. Oxidative stress parameters Lipid peroxidation (LPO), Superoxide Dismutase (SOD) and Reduced Glutathione (GSH) were analyzed by the methods of Placer et al. (1966), Nishikimi et al. (1972) and Hafeman et al. (1974).  Body condition score (BCS) assessment was done as per the methods of Ferguson et al. (1994) and milk yield was also recorded. Data was analysed by one way ANOVA and independent sample t-test by using SPSS software.

Results and Discussion

The mean Hb values were in the normal range and no significant effect was observed of SCK on the mean Hb values (Table 1).

Table1: Haematological indices in crossbred cows affected with subclinical ketosis (Mean± S.E.)

Parameters Period Healthy (n=18) SCK (n=22)
FOD 10.87±0.38x1 11±0.34w1
Hb(g/dl) CUD 11.08±0.41x1 10.01±0.34yx1
Fresh 10.72±0.34x1 9.16±0.33zx2
FOD 34.55±1.41x1 34.32±1.11w1
PCV (%) CUD 34.95±1.25x1 30.90±1.12yx1
Fresh 33.78±1.08x1 29.23±1.23zx2
TEC FOD 8.12±0.50x1 5.91±0.17x2
(x106/μl) CUD 7.92±0.49x1 5.90±0.17x2
Fresh 7.13±0.48x1 5.49±0.25x1
TLC FOD 7.84±0.32x1 8.22±0.38x1
(x103/μl) CUD 8.07±0.46x1 8.80±0.42x1
Fresh 7.71±0.43x1 9.37±0.76x1

Values bearing different superscript (w, x, y, z) in a column differ significantly

Values bearing different superscript (1, 2) in between healthy and SCK cows differ significantly

However, a significant (p<0.05) decrease was noted from FOD upto the fresh period in the Hb values  in SCK cows, whereas in  comparison to the healthy animals, significantly lower values were observed for  Hb and PCV values in SCK cows during fresh period. Hematological parameter analysis revealed that all the hematological parameters (Hb, PCV, TEC and TLC) were in the normal range (Radostits et al., 2007), There are various studies reporting different results regarding the effect of SCK on hematological parameters, Marutsova et al. (2015) reported a increase in the Hb, PCV and TEC levels in clinical and subclinical ketotis cows, where as Belic et al. (2010) reported a decrease in the hemoglobin level and RBC counts in ketotic cows, and cited that the possible reason for the decrease is due to erythropenia that is accompanied by anisocytosis and poikilocytosis.

The overall mean TEC levels recorded during the FOD, CUD and fresh period, were within the normal physiological range, and in comparison to healthy animals significantly (p<0.05) lower value was observed for overall mean TEC during FOD and CUD period in the SCK cows (Table 1). However a non significant increase was observed in the mean TLC values from the FOD period upto the fresh period in SCK cows and non significant difference was recorded in comparison to the healthy animals (Table 1).

Similar results were also reported by Marutsova et al. (2015) who in his respective study reported an increase in the TLC count in SCK cows in comparison to control cows, this increase in TLC in SCK cows could be due to the increased neutrophil and lymphocyte counts reported in lactating cows in response to the enhanced lipomobilisation, ketogenesis and hypoglycemia (Belic et al., 2010), increased stress and accompanying levels of glucocorticoids (cortisol) (Burton et al., 2005) and also due to the immunosuppression caused by the production of high ketone bodies concentrations.

Biochemical Studies

Biochemical parameter analysis revealed a significant decrease in the TPP and glucose levels from the FOD upto the fresh period in SCK cows and in comparison to healthy cows significantly lower glucose levels (lower than the normal range) were observed in SCK cows during the fresh period. However, in SCK cows a significant increase was recorded from the FOD upto the fresh period in the plasma BHBA and NEFA values and significantly higher levels were recorded for the plasma creatinine, BHBA and NEFA levels in SCK cows in comparison to the control cows (Table 2).

Table 2: Biochemical profile in crossbred cows affected with subclinical ketosis (Mean± S.E.)

Parameters Period Healthy (n=18) SCK (n=22)
TPP (g/dl) FOD 6.62±0.19x1 7.40±0.19wx2
CUD 6.94±0.35x1 6.95±0.17yx1
Fresh 6.92±0.28x1 6.37±0.16z1
Albumin (g/dl) FOD 3.05±0.26x1 2.72±0.13x1
CUD 2.81±0.17x1 2.66±0.09x1
Fresh 2.70±0.12x1 2.60±0.14x1
PUN (mg/dl) FOD 9.61±1.01x1 8.27±0.43x1
CUD 9.88±1.03x1 9.63±0.62x1
Fresh 10.50±1.12x1 10.22±1.06x1
Creatinine (mg/dl) FOD 0.98±0.08x1 1.53±0.20x2
CUD 1.07±0.13x1 1.46±0.12x2
Fresh 1.11±0.19x1 1.34±0.11x1
Glucose (mg/dl) FOD 67.11±3.86x1 63.81±3.22wx1
CUD 75.72±3.62x1 65.50±4.87x1
Fresh 68.11±4.03x1 43.23±1.77z2
BHBA (mmol/L) FOD 0.37±0.16w1 0.55±0.03x2
CUD 0.52±0.0.47yx1 0.76±0.04y2
Fresh 0.57±0.04wx1 1.39±0.02z2
NEFA (mmol/L) FOD 0.14±0.02w1 0.20±0.02x1
CUD 0.27±0.03yx1 0.43±0.07y1
Fresh 0.26±0.02zx1 0.68±0.05z2

* Values bearing different superscript (w, x, y, z) in a column differ significantly

* Values bearing different superscript (1, 2) in between healthy and SCK cows differ significantly

Similar results were also reported by Li et al. (2016) where the authors also reported a decrease in blood glucose in cows with subclinical ketosis. This hypoglycemia recorded in SCK cows was ascribed due to the decreased gluconeogenesis (Goff and Horst, 1997).  It has been seen that in the SCK cows due to the decrease in glucose output by the liver there is decrease in the blood glucose concentrations, due to which there is decreased insulin secretion, which, in turn, leads to increased lipid mobilization from adipose tissue and increased rate of hepatic fatty acid uptake and ketogenesis (Grummer, 1995). Blood BHBA is generally regarded as gold standard test for diagnosis of subclinical ketosis in cows, as BHBA is being more stable ketone body than acetone or acetoacetate. A number of studies (Ribeiro et al., 2013; Li et al., 2016) have also reported results similar to the present findings where they classified a cow affected with SCK on the basis of blood BHBA level above 1.2 mmol/L.  This increase in NEFA and BHBA in cows with SCK was credited to the increased fat mobilization from the adipose tissue during the early lactation period to support the negative energy balance, when blood glucose levels were low due to the initiation of milk production, resulting in increased production of acetyl-CoA, which resulted in increased production of ketone bodies (Allen and Piantoni, 2013).

As regards to other biochemical parameters, a significant decrease was recorded in the TPP levels from the FOD upto the fresh period, where as a non significant decrease was noted for all the other parameters in the SCK cows (albumin, PUN and creatinine). Similar to our study Djokovic et al. (2013) and Li et al. (2016) also reported a significant decrease in total plasma proteins and a non significant decrease in albumin, urea, triglycerides and total bilirubin levels during early pregnancy in cows suffering from subclinical ketosis, as the plasma levels of the glucose, TPP, albumin and urea are the  indicators of hepatic functionality (Bobe et al., 2004) and decrease in their concentration in the plasma  suggests fat infiltration into the liver (Djokovic et al., 2013). Similarly Gonzales et al. (2011) also observed a decrease in plasma albumin, total proteins and urea in cows with subclinical ketosis. However, Li et al. (2011) recorded decrease in the renal function in cows suffering with subclinical ketosis, where as in our study non significant differences was seen in the PUN and creatinine levels.

Plasma mineral concentration evaluation revealed a significant decrease in the Ca, Na and Zn levels and non significant decrease in Pi, K and Cu Zn, where as a non significant increase was seen in Mg and Fe levels in SCK cows. However, in between the SCK and the healthy cows non significant differences were observed for all other parameters (Mg, K, Cu, Fe and Zn) and non significant changes were recorded from the FOD upto the fresh period for all the parameters  (Table 3). During the early milking period when there is higher demand of Ca for the increased milk production, negative energy balance (NEB) develops in cows suffering from SCK due to which there is decrease in Ca concentration (Ribeiro et al., 2013).

 

 

Table 3: Plasma mineral concentrations in crossbred cows affected with subclinical ketosis (Mean± S.E.)

Parameters Period Healthy (n=18) SCK (n=22)
Ca (mg/dl) FOD 9.89±0.52wy1 10.29±0.43xw1
CUD 9.38±0.52xy1 9.62±0.33xy1
Fresh 8.38±0.42xz1 8.40±0.32z1
Mg (mg/dl) FOD 3.77±0.22x1 3.55±0.14x1
CUD 3.66±0.34x1 3.47±0.15x1
Fresh 3.30±0.20x1 3.74±0.28x1
Pi (mg/dl) FOD 4.94±0.19x1 4.82±0.22x1
CUD 4.92±0.21x1 4.43±0.25x1
Fresh 4.39±0.17x1 4.37±0.15x1
Na (mmol/l) FOD 122.53±1.77wx1 126.37±2.85wx1
CUD 121.24±4.49xy1 120.52±1.72xy1
Fresh 113.14±2.39yz1 112.49±1.92zy1
K (mmol/l) FOD 5.50±0.21xw1 5.79±0.23x1
CUD 4.83±0.31xy1 5.30±0.14x1
Fresh 4.69±0.15zy1 4.85±0.20x1
Cu (ppm) FOD 0.86±0.12x1 0.95±0.09x1
CUD 0.83±0.14x1 0.84±0.06x1
Fresh 0.70±0.07x1 0.93±0.10x1
Fe (ppm) FOD 3.01±0.34x1 2.88±0.44x1
CUD 3.29±0.41x1 2.96±0.43x1
Fresh 3.34±0.37x1 3.15±1.40x1
Zn (ppm) FOD 1.24±0.11wx1 1.09±0.10wx1
CUD 1.06±0.88yx1 1.10±0.18yx1
Fresh 0.71±0.0627z1 0.69±0.07z1

Values bearing different superscript (w, x, y, z) in a column differ significantly

 Values bearing different superscript (1, 2) in between healthy and SCK cows differ significantly

 

Additionally, another reason for the decrease in Ca level in SCK cows could be due to the increased loss of base in the urine to compensate for the acidosis (Radostits et al., 2007). Similar to our study Karimi et al. (2015) reported a decrease in the cooper and zinc levels along with a increase in the LPO levels during the first week after calving in SCK cows indicating the role of minerals in the antioxidant defence. Increase in the level of magnesium observed in hypocalcemic cows from FOD upto fresh period in our study might be due to the shift of magnesium from the intracellular to the extracellular compartment which might have caused transient increase in the Mg levels, another reason for this increase could be due to the increased tubular reabsorption of magnesium due to the elevated levels of serum PTH during the transition period on kidneys (Riond et al., 1995). Several contributing factors have been found responsible for the lower Pi concentration in SCK cows, firstly with the increase in milk production, and due to the development of NEB, most of the phosphorus from the ingested amount is transferred for milk synthesis, (Valk et al., 2002) secondly with the higher PTH levels induced by low plasma Ca in the SCK cows, there is increases in the urinary loss of P and finally due to decrease in nutrient intake there is decrease in the amount of P absorption from the intestinal tract, resulting in decrease in the plasma P levels. Similarly Zhang et al. (2010) reported no significant effect on plasma Na and K levels in cows with subclinical ketosis. However, a significant decrease was recorded in the mean plasma Zn levels in SCK cows as compared to the healthy cows. A significant lower level for the BCS was observed in the SCK cows in comparison to healthy cows during the fresh period and a significant decrease was also recorded from the FOD upto the fresh period in both the groups and similarly a significant decrease was also noted in the milk yield in the SCK cows in comparison to healthy cows (Table 4).

Table 4: Body condition score and milk yield in crossbred cows affected with subclinical ketosis (Mean±S.E.)

Parameters Healthy (n=18) SCK (n=22)
 

BCS

FOD 3.69±0.03w1 3.70±0.04x1
CUD 3.40±0.04xw1 3.27±0.05y1
Fresh 3.08±0.041z 2.84±0.072z
Milk Yield 26.44±0.871 18.54±1.112

Values bearing different superscript (w, x, y, z) in a column differ significantly

Values bearing different superscript (1, 2) in between healthy and SCK cows differ significantly

Generally, body condition reflects the amount of sub cutaneous body fat of cows (Ferguson et al., 1994) and BCS is usually done during dry and early lactation periods, because of the links with subsequent performance. Similar to the present study, Ribeiro et al. (2013) revealed that cows with subclinical disease had higher BCS at first 7 days of calving, but looses more BCS during the 7-35 days in milk. Similarly Bernabucci et al. (2005) in his study stated that cows with higher body condition score (BCS) and greater BCS loss were particularly sensitive to oxidative stress. Similarly lower milk yield was recorded in SCK cows, however, the higher standard error (S.E.) value observed in SCK cows was due to a cow with milk yield 38 litre was also found suffering from SCK during early milking, which was ascribed due to the higher milk yield resulting in increased fat mobilization and greater development of ketone bodies (Lean et al., 1992). Decreased milk yield observed in early lactation might be due to the hypoglycemia observed in SCK which causes drop in lactose synthesis and hence reduced milk production (Simensen et al., 1990).

A significant increase was seen in the lipid peroxidation (LPO) levels in the SCK cows from the FOD to fresh period, where as in comparison to healthy cows, significantly higher levels were seen for the LPO levels in SCK cows in comparison to control cows. This significant increase in the lipid peroxidation especially after calving could be due to the increased metabolic demands imposed on the cow by colostrum production and the onset of lactation that far exceeded the demands of the fetus leading to subclinical ketosis. Similar results were also reported by Karimi et al. (2015) and Yi et al. (2016) who in their respective studies reported an increase in the LPO levels in cows suffering from subclinical ketosis. Similarly, a significant decrease was recorded from the FOD upto the fresh period for the SOD and GSH levels in the SCK cows, where as in comparison to healthy cows significantly lower levels were recorded for the SOD during the CUD and fresh period, where as no difference was noted in the GSH levels in the healthy and SCK cows (Table 5).

Table 5: Oxidative stress markers in healthy and SCK cows (Mean± SE)

Parameters   Healthy SCK
LPO FOD 221.59±4.75wx1 238.72±12.37x1
CUD 255.58±7.66 wy1 309.56±13.09y2
Fresh 309.51±8.74z1 365.80±16.21z2
SOD FOD 94.11±5.50x1 76.88±2.99 x 2
CUD 77.82±3.23y1 63.29±2.52y2
Fresh 59.35±3.67z1 48.36±2.61z2
GSH FOD 2.98±0.151x 2.65±0.091wx
CUD 2.48±0.101y 2.34±0.091wy
Fresh 2±0.091z 2.08±0.101z

Values bearing different superscript (w, x, y, z) in a column differ significantly

Values bearing different superscript (1, 2) in between healthy and SCK cows differ significantly

Similarly a decrease was noted in the SOD and GSH levels in SCK cows depicting a decrease in the antioxidant defence in the body during subclinical ketosis. As is well known, SOD is a Cu/Zn-dependent enzyme and erythrocyte GSH-Px is a Se-dependent enzyme, so reduction in zinc and copper availability in the early postpartum period (Muehlenbein et al., 2001) of dairy cows might explain the reduction of SOD activity during early lactation period. Reduced levels of GSH were observed in the present study, which might be due to the decreased production or increased depletion. Similarly Sharma et al. (2011), also recorded a significant depletion in the blood GSH levels due to the increased production of ROM during the early lactation as compared to the advanced pregnancy, along with a significant positive correlation between the GSH and LPO during the early lactation period.

The results of the present study showed higher BHBA and NEFA levels are very important indicators in the diagnosis of subclinical ketosis and also these cows are more prone to increased oxidative stress. And also a significant effect was seen of sub clinical ketosis on milk yield, body condition scoring and the metabolic parameters.

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