The influence of condition on the metabolic profile of Czech Fleckvieh cows in the perinatal period

The objective of the study was to determine the influence of Czech Fleckvieh cow‘s condition before parturition on chosen blood biochemical indices in perinatal period and at the beginning of lactation. The study was conducted on 38 multiparous cows 3-14 days prepartum. The cows were selected with respect to body condition score (BCS): BCS>4 points (group I, n=18) and BCS<4 points (group II, n=20). Blood was collected at 3-14 and 1-2 days prepartum and 1-2, 21 days postpartum and analyzed for lipid-carbohydrate, protein parameters and enzymatic activity. Cow‘s condition, milk yield and composition were also estimated. Changes in Czech Fleckvieh cow‘s condition in a transition period do not have any clear influence on milk yield and composition. An excessive BCS before calving leads to its higher decrease at the beginning of lactation and may indirectly point to a higher energy deficiency in the first period of lactation. It is confirmed by glucose concentration in blood serum decrease (P<0.01) in 3rd week of lactation when compared to cows with a proper condition before calving (3.39 vs. 2.45 mmol/l). Overnormative condition before calving also negatively influences the content of triacylglycerols, alkaline phosphatase and total bilirubin in blood serum in the first days postpartum. TAG transport from liver to blood is impaired at the beginning of lactation in cows with high condition prepartum. The study demonstrated that in Czech Fleckvieh dairy type cows intensified lipolysis and ketogenesis in perinatal period do not take place pointing to high adaptation possibilities of their metabolism.


Introduction
Dairy cows are more susceptible to various diseases in the postpartum period when compared to other production periods, due to the negative energy balance, weakened appetite and increasing yield.That is a period particularly favourable for metabolic disorders (Rajala-Schultz et al. 1999).Difficult parturition and problems with fertilisation are more probable to occur in excessively fat cows (Roche et al. 2007).Milk performance in a subsequent lactation depends usually on the proper body condition score (BCS) of cows in the last 100 days of the current lactation (Rodenburg 1992).
The diagnosis of metabolic disorders in herds of cows is based on a whole set of diagnostic examinations, where the determination of biochemical blood indices is an important element (Oetzel 2004, Mordak & Nicpoń 2006).Preventive-diagnostics programmes also include laboratory analyses of milk, urine, rumen content, and of feed doses (Studer 1998).They aim to eliminate the risk for animal health just before the occurrence of clinical symptoms of disease, reproduction disorders and a decrease in milk yield.Periodical diagnostic monitoring of the herd is of a great significance in the prevention of metabolic disorders and allows additional economic indices in dairy cows breeding to be obtained (Quiroz-Rocha et al. 2009).
In the assessment of the results of laboratory blood analyses, the reference value characteristics for the given cattle population (breed, direction and scale of production) should be taken into consideration.Physiological standards are usually contained within a wide range of values, and as a result, comparisons to them are of a lower diagnostic value, especially in the case of disorders of a subclinical character.The determination of particular biochemical parameters of blood in clinically healthy cows of a given breed with respect to physiological state, performance direction and feeding may provide important information when interpreting results in animals exhibiting pathological symptoms.The assessment of particular biochemical parameters in blood of cows of the Czech Fleckvieh breed of dairy type is justified due to the large abundance of breeding in numerous countries.The study concerning only an assessment of lipid profile can be found in the literature (Borucka-Jastrzębska et al. 2007).Also, studies on the Montbeliard breed have been conducted (Ferlay et al. 2006).The aim of the study was to determine the influence of the condition of Czech Fleckvieh cows (dairy type) before the parturition on chosen biochemical indices of blood in the perinatal period and at the beginning of lactation.The study also aimed at an assessment of an excessive condition of cows in the terminal dry-off period on the level of analysed parameters in blood.

Animals
The study was conducted on a Czech Fleckvieh cow farm owned by Proagro Radešínská Svratka (Czech Republic).The herd was free from infectious and invasive diseases.The number of cows in the herd was 330 individuals with an average yield of 6 990 kg of milk, 3.8 % fat and 3.49 % protein.Animals in the perinatal period were kept in a nursery on a deep litter in collective pens (about 15 individuals in each).In the 7th day after parturition, they were moved to a loose barn.Feeding was based on Total Mixed Ration (TMR) doses.Dry cows received about 15 kg of TMR dose and straw ad libitum, while lactating cows 50.3 kg TMR daily.The diets were balanced with regard to the energy content, crude protein and minerals.The dietary nutrient content for dairy cows is given in Table 1.The composition of TMR dose applied in the study stayed intact.

Experimental design, blood sampling and analysis
The study was conducted on 38 multiparous cows in the period 3-14 days prepartum, and selected with respect to the condition, i.e. cows of BCS>4 pts.(group I, n=18) and cows of BCS<4 pts.(group II, n=20).The method of assessment was consistent with the recommendations of the European Association of Fleckvieh Cattle Breeders (1=thin cow, 5=over conditioned cow).
The assessment was done by the same person 3-14 days prepartum (start of the experiment), 1-2 days prepartum, 1-2 days postpartum, and in the 3rd week of lactation.The animals were clinically healthy, and aged 4-8 years.
The blood for analysis was collected from an external vein into disposable test tubes without anticoagulant addition in the morning before feed application between 3rd and 14th days prepartum (38 cows), and then on the days when the condition was assessed: 1-2 days prepartum, 1-2 days postpartum and in the 3rd week of lactation.
Cows were milked twice daily in a double-12 parallel milking parlour and milk yield was recorded from which a weekly average for each cow was calculated for weeks 1-4 postpartum.Representative milk samples were analysed for protein, fat, lactose using a Bentley 150 -Infrared Milk Analyzer (Bentley Instruments, Chaska, USA).Sample milking was conducted in the 3rd week of lactation (end of collected the blood).Body mass was determined before calving and at the 3rd week of lactation.

Statistical analysis
The results of the study were subject to statistical analysis using the SAS statistical packet (SAS 2009) based on a linear model from GLM procedure that respected: where x ij is the the observed values of an analysed parameter, µ is the mean of the parameter in a population, s i is the the effect of i-th -year of cows' birth and e ij is the an error.
Significance of differences between mean values in both groups was determined using Duncan's test.

Results
According to the methodology it was assumed the condition of cows from group I was higher before the parturition (4.23 pts.) when compared to group II (3.76 pts.).The condition of cows tended to decrease up to the 3rd week of lactation (Figure 1).Before calving the body mass of cows with a high BCS score was significantly (P<0.01)higher.A significant (P<0.01)decrease in body mass from the 3rd week of lactation when compared to the beginning of the study was observed in both groups (Table 2).
The decrease in condition in group I (P<0.01) was higher when compared to group II (P<0.05).No significant differences between the groups in terms of the yield and composition of milk were observed (Table 2).**significance of differences between the groups within collection with P≤0.01, A,B Significant differences are marked with different letters between the groups P≤0.01 a/A: significance of differences between particular days of assessment with P≤0.05/P≤0.01Mean values for selected serum lipid-carbohydrate parameters are presented in Table 3.A slight increase in glucose concentration in blood serum in group I (high condition) was noted directly after parturition (1-2 days postpartum).This could have resulted from physiological postpartum stress and higher secretion of glicocorticoids that increase the glucose level.In the 3rd week of lactation the level of glucose was subject to a significant (P<0.01)decrease in group I when compared to other studied periods.Differences between the groups were significant (P<0.01).Before the parturition (1-2 days) the concentration of BHBA in blood in group II was lower (P<0.05)when compared to group I. Up to the 3rd week of lactation a statistically significant increase in BHBA was noted in group II.An increase (P<0.05) in NEFA concentration in the prepartum period was observed in group II.A slight increase (P<0.05) in NEFA level was noted postpartum in cows from group I, characterised by an overnormative condition prepartum (Table 3).In the case of group II, the concentration of NEFA was subject to small changes up to the 3rd week of lactation.The level of triacylglycerols decreased significantly (P<0.01) in the 3rd week of lactation in group I.In days 1-2 and in the 3rd week postpartum, the concentration of TAG in blood serum in group I was significantly (P<0.01)lower when compared to group II.In the group of cows with an overnormative condition prepartum (group I) a decrease (P<0.05) in cholesterol concentration in blood serum was observed directly postpartum (1-2 day) (Table 3).In the 3rd week of lactation, an increase in cholesterol level was distinct (P<0.01) in both groups.*significance of differences between groups within sampling with P≤0.05, **significance of differences between groups within sampling with P≤0.01, a,b Significance of differences marked with different letters between particular days of assessment in a group with P≤0.05, A,B,C,D,E,F Significance of differences marked with different letters between particular days of assessment in a group with P≤0.01 No significant differences in AST and ALT activity between the groups before the parturition were observed (Table 4).An increase in AST activity up to 79.13 U/l was noted at 1-2 days postpartum in group I.A lower increase was observed in cows from group II, and differences between the groups were significant (P<0.05).In the 3rd week of lactation, an increase in ALT activity (P<0.01) was observed in both groups.On the day of the beginning of the study higher (P<0.05)activity of GT was noted in cows from group I, however the activity of that enzyme postpartum was subject to decrease (P<0.05).Similarly, in the period 3-14 days prepartum, higher (P<0.01)activity of ALP was noted in group I, and it decreased postpartum (P<0.01).A higher concentration of total bilirubin (5.11 μmol/l) was observed prepartum in group I (P<0.01).A similar relationship was noted in the case of GGT and ALP activity.A significant increase in bilirubin concentration (P<0.01) was observed postpartum (1-2 days) in group II (5.18 μmol/l).This value was subject to decrease in subsequent sampling (P<0.01) to the level of 3.32 μmol/l.*significance of differences between groups within sampling with P≤0.05, **significance of differences between groups within sampling with P≤0.01, a,b Significance of differences marked with different letters between particular days of assessment in a group with P≤0.05, A,B,C,D,E,F Significance of differences marked with different letters between particular days of assessment in a group with P≤0.01 *significance of differences between groups within sampling with P≤0.05; **significance of differences between groups within sampling with P≤0.01; a,b Significance of differences marked with different letters between particular days of assessment in a group with P≤0.05.A,B,C,D,E,F Significance of differences marked with different letters between particular days of assessment in a group with P≤0.01 Higher (P<0.01)concentration of total protein was observed in blood serum of cows from group I in the period 1-2 days prepartum, when compared to group II (Table 5).Up to the 3rd week of lactation, the content of TP in blood of cows from group II increased (P<0.01).A higher content of albumins (similar to the situation with TP) was noted prepartum in group I, and it tended to decrease successively (P<0.01) up to the 3rd week of lactation.When compared to the beginning of the study, the content of urea in group I was subject to decrease to 1-2 days postpartum, and then it increased (P<0.01).The content of urea in blood serum in group II in 3rd week of lactation reached the same value as in the case of group I (5.73 mmol/l).

Discussion
The body condition score of cows at calving and its decrease in the period of early lactation were connected with a risk of metabolic diseases (Heuer et al. 1999), deterioration in fertility and milk yield (Heuer et al. 1999, Reksen et al. 2002).The decrease in the condition of Holstein-Friesian cows (Hachenberg et al. 2007) and Czech Fleckvieh (Jílek et al. 2008) reflects the degree of energy deficiency, since mobilization of body fat is associated with the negative energy balance postpartum.
It was not proved in the case of Czech Fleckvieh cows that a higher BCS value prepartum was connected with a higher decrease of BCS postpartum, without any influence on reproduction (Jílek et al. 2008).In the present study, the decrease in the BCS condition of cows with a high score prepartum was significant (P<0.01) up to the 3rd week.It was, however, the short period postpartum.Roche et al. (2007) stated that the BCS at the lowest point was positively correlated with BCS at calving.The degree of BCS condition decrease as observed in the present study in both groups was also observed in the case of a large population of Czech Fleckvieh cows (Jílek et al. 2008).Cows of the Simmental breed with the condition <3.5 pts. in the 1st week postpartum are characterized by the highest milk yield in the first 5 months of lactation (Jílek et al. 2008).In the present study, cows with an overnormative condition prepartum were characterized by a slightly higher condition in that period, that could have been connected to the changes in milk yield in the case of longer study.
The decrease in glucose level in the first weeks postpartum is a demonstration of metabolic adaptation to the start of lactation (Studziński et al. 2003, Dann et al. 2005).The occurrence of a negative energy balance may be connected with a decrease in the glucose content in blood to a level even over 1/3, and with an increased level of ketone compounds and FFA (Studziński et al. 2003).In the present study cows in which BCS condition was high prepartum, a significant lower (P<0.01)concentration of glucose in blood was observed in the 3rd week of lactation when compared to cows with a lower condition prepartum, i.e. 2.45 vs. 3.39 mmol/l, however, with a lack of intensified lipolysis and ketogenesis.The concentration of BHBA in blood serum during the whole research period was considerably below the boundary value of 1.4 mmol/l in both groups (Carrier et al. 2003, Oetzel 2004, Kupczyński et al. 2005).Any risk of subclinical ketosis was noted during the research period.The observed value of BHBA before parturition (108 days) was at the level of 218-884 µmol/l (Quiroz-Rocha et al. 2009).The values observed in the present study were from 0.28 to 0.47 mmol/l on average, and were higher with higher condition.In the case of highyielding cows of the Holstein-Friesian breed in turn, there is a risk of an excessive lipolysis in the first weeks of lactation, even proper feeding and the application of glucogenic precursors (Kupczyński et al. 2005).
The proper level of NFA in blood serum in dairy cows from 14 to 2 days prepartum should be lower than 0.4 mmol/l (Oetzel 2004).In the present study on Czech Fleckvieh cows the content of NEFA in that period was 0.41 mmol/l (group I) and 0.28 mmol/l (group II).In spite of this fact, no excessive lipolysis was noted postpartum.In the study with crossbreds of beef breeds, it was observed that cows with a high condition at calving were characterised by lower NEFA concentration in blood (Vizcarra et al. 1998).The differences were not significant in the present study.The decrease in fodder intake observed in the perinatal period (Ferlay et al. 2006) causes an intensified lipolysis of the reserve fat (Studer et al. 1998).The manifestation of these changes is an increase in NEFA level in blood directly before calving, reaching a peak 3-5 days postpartum.The return to their low concentration takes place about 14-21 days postpartum (Vazquez-Añon et al. 1994).In high yielding cows of PHF breed, an intensified lipolysis may be maintained up to the 3rd week of lactation (Kupczyński et al. 2005).The yield of analysed Czech Fleckviech cows in that period was 37.4 kg of milk (group I), and 36.2 kg (group II), however, no overnormative increase of NEFA in blood serum was not observed.Cows of the Montbéliarde breed grazed on pasture for 6 weeks were characterised by an increased concentration of NEFA and BHBA that points to insufficient nutrition (Ferlay et al. 2006).Breed differences were also demonstrated in another study (French et al. 2006).In the period 3 days prepartum to 1 day postpartum, the level of FFA for Holstein and Jersey breeds was 775 vs. 521 μEq/L, respectively (French et al. 2006).Body condition score at parturition and postpartum nutrition influence the occurrence of luteal activity and the concentration of glucose, insulin, and nonesterified fatty acids in the plasma of primiparous beef cows (Vizcarra et al. 1998).The highest concentration of FFA in blood of Holstein-Friesian cows was noted in the 1st week of lactation (Hachenberg et al. 2007) pointing to the fact that the concentration of FFA >0.5 mol/l in that period is a significant criterion limiting the possibilities of metabolic adaptation post calving.Beef cows with a BCS lower at parturition had a decrease in circulating NEFA and an increase in serum GH (Lake et al. 2006).Even postpartum dietary lipid supplementation does not appear to influence metabolic signals associated with nutrient partitioning in beef cows during early lactation.TAG concentration in blood tends to decrease with hyperketonemia severity (Marczuk & Filar 2003).In the present study, TAG content in the 3rd week of lactation in cows that had a high prepartum condition decreased to a lower limit of reference values (Meyer & Harvey 1998).An increase in BHBA was not observed.A higher level of triglycerides and similar of total cholesterol when compared to the present study was observed in the case of dairy type Simmental cows (Borucka-Jastrzębska et al. 2007).The reduction of serum cholesterol is related to the switch in synthesis of lipoproteins from the hepatocytes (Quiroz-Rocha et al. 2009).An increase in cholesterol levels after parturition, irrespective of the condition of cows, was observed in the present study.
Parturition and starting lactation influence the activity of AST in blood serum of cows of a condition higher before the parturition (group I).Marczuk & Filar (2003) point out the special usefulness of the determination of GLDH, GGT, AST activity and bilirubin concentration for the recognition of subclinical impairments and liver disorders like fatty liver.The changes observed in the present study were not intensified, which proves the proper functioning of the liver.The observed activities of enzymes in the blood in both groups did not diverge from values observed in cows of the Holstein-Friesian breed (Mordak & Nicpoń 2006).
The concentration of total bilirubin during the whole research period did not exceed the reference values (Meyer and Harvey 1998).A higher concentration of bilirubin prepartum was noted in cows with higher BCS; however that did not influence its content postpartum.The value of that index in cows with subclinical ketosis was at the level of 7.27 μmol/l (Nowakowski 2008), and in cows with subclinical form of fatty liver in 1-2 weeks postpartum it was 14.2 μmol/l (Marczuk & Filar 2003).The results of the present study demonstrate a lower risk of hyperbilirubinemia incidence in Simmental cows when compared to Holstein-Friesian ones.
The concentration of total protein and albumins in blood serum in the perinatal period was within the reference values (Meyer & Harvey 1998).The fluctuations observed in the perinatal period were small; however, they were confirmed statistically.Levels of urea similar to that observed in the present study were found in cows of the Montbeliard breed grazed on pasture (Ferlay et al. 2006).In the present study, a decrease in urea level (P<0.01)1-2 days postpartum was observed in cows with a higher condition prepartum.Quiroz-Rocha et al. (2009) suggest that reduced dry matter intake might cause a drop in ammonia absorption, causing a shift of urea being recycled in the rumen, which may explain the lower serum concentrations of urea in the postpartum period.
It should be stated in summary that changes in Czech Fleckvieh breed cows in the transition period do not clearly influence milk yield and composition.An excessive BCS condition before calving leads to its higher decrease at the beginning of lactation what may indirectly point to higher energy deficiency.It is confirmed by a decrease (P<0.01) in glucose concentration in blood serum in the 3rd week of lactation when compared to cows with a proper condition before calving (3.39 vs. 2.45 mmol/l).Overnormative condition of cows before calving also negatively influences the content of TAG, ALP and Total bilirubin concentration in blood serum on the first days after calving.In cows with high condition before calving, the transport of TAG from liver to blood is impaired at the beginning of lactation.The present study demonstrates that in the Czech Fleckvieh breed of dairy type, intensified lipolysis and ketogenesis in the perinatal period (1-2 prepartum to 1-2 days postpartum) do not take place, pointing to high adaptation possibilities of the metabolism.Directed biochemical analysis of blood in the perinatal period allows metabolic disorders in subclinical form to be detected, thus avoiding the risk of the development of full-blown disease symptoms.

Figure 1
Figure 1 Estimation of cow condition by BCS method (points)

Table 2
Milk yield, milk composition and body weight (±s)

Table 3
Means for serum blood metabolite concentrations prepartum and postpartum (±s)