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Research Article
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Studies on the Relationship Between Sub-Clinical Ketosis and Liver Injuries Within the First Two Months of Lactation Period in High Producing
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S. Nazifi,
M. Mohebbi Fani,
E. Rowghani
and
M.R. Behbood
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ABSTRACT
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The relationship between Sub-Clinical Ketosis (SCK)
and liver injuries within the first two months of lactation in three commercial
dairy herds with rather constant routines in management and nutrition
was studied. A total of 77 cows (38 cows in the first and 39 cows in the
second months of lactation) were sampled for blood. The serum concentrations
of glucose, beta-hydroxybutyrate (BHB), nonesterified fatty acid (NEFA),
aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), cholesterol,
triglyceride and VLDL-cholesterol were measured at 30 and 60 days after
calving. Sub-clinical ketosis was considered in cows with serum concentration
of BHB>1000 μmol L-1. The concentration of serum glucose
in cows with SCK was significantly (p<0.05) lower than healthy cows
after 30 days of calving. However, the concentrations of serum BHB, NEFA,
triglyceride and VLDL-Cholesterol in SCK cows were significantly higher
(p<0.05) than the healthy cows. In second month of lactation, the concentrations
of serum BHB and NEFA in SCK cows were significantly higher than the healthy
cows. The concentration of serum BHB, NEFA, triglyceride and VLDL-cholesterol
in SCK cows were significantly higher (p<0.05) than the healthy cows
at 30 and 60 days postpartum periods. In the first and second months of
lactation, a positive significant correlation was observed between serum
glucose and GGT (R = 0.409, p<0.05) in the healthy cows. However, significant
correlations were observed between serum glucose and cholesterol (R =
0.403, p<0.05) and GGT and cholesterol (R = 0.388, p<0.05) in cows
with SCK. Hepatic injuries were not observed in cows with SCK. In spite
of negative energy balance in the first and second months of lactation,
liver function tests were normal. The results of this study showed that
the concentration of serum BHB and NEFA of SCK cows within the first two
months of lactation was significantly higher than healthy cows, possibly
due to higher energy demands of cows at this stage.
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How
to cite this article:
S. Nazifi, M. Mohebbi Fani, E. Rowghani and M.R. Behbood, 2008. Studies on the Relationship Between Sub-Clinical Ketosis and Liver Injuries Within the First Two Months of Lactation Period in High Producing. International Journal of Dairy Science, 3: 29-35. DOI: 10.3923/ijds.2008.29.35 URL: https://scialert.net/abstract/?doi=ijds.2008.29.35
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INTRODUCTION
Health and performance management systems for dairy cattle need to focus
on early identification and subsequent prevention of production diseases
such as clinical and sub-clinical ketosis (Ingvartsen et al., 2003)
by treating individual cows or to improve the herd diet (Enjalbert et
al., 2001). The current convention is to maximize DMI and energy intake
prepartum and minimize the drop in DMI as parturition approaches (Mashek
and Grummer, 2003). Dramatic increases in energy requirements during late
gestation and early lactation superimposed an animal with a profound drop
in DMI before calving and make the dairy cow highly susceptible to the
metabolic diseases e.g., ketosis and hepatic lipidosis (Osborne, 2003).
The animal attempts to supply the needs for milk production by drawing
on body fat reserves. This release of free fatty acids results in the
production of the major ketone bodies, acetone, acetoacetate and beta-hydroxybutyrate
(BHB) (Dann et al., 2005; Padilla et al., 2005). These compounds
are important source of energy when carbohydrate levels are reduced (Duffield,
2000). Sub-Clinical Ketosis (SCK) is defined as elevated concentrations
of circulating ketone bodies in the absence of clinical signs of ketosis
(Duffield, 2000). Cows are at risk of sub-clinical ketosis within the
first two months postpartum (Duffield, 2000). SCK can affect milk production
(Rajala-Schultz et al., 1999; McLauren et al., 2006), reproduction
(Walsh et al., 2007a, b), increased frequency of left displaced
abomasum (Grohn, 2000; LeBlanc et al., 2005) and decreased in nonspecific
immunity (Sartorell et al., 2000). Reported overall incidence rates
of SCK range from 6.9 to 14.1% in the first 2 months of lactation (Duffield
et al., 1997) although prevalence as high as 34% has been reported
(Duffield, 2000).
Test results could be used on a herd basis to determine the level of
SCK and indicate the necessity for further investigations and management
improvements. Blood glucose and BHB concentrations have been used as biological
indicators reflecting the status of the dairy cow.
The gold standard diagnostic test for SCK is the measurement of BHB in
serum or plasma because of its stability (Duffield, 2000; Herdt, 2000;
Oetzel, 2004). There have been many thresholds that have been used to
distinguish between healthy cows and cows with SCK (Duffield et al.,
1998; Geishauser et al., 1998). However, BHB levels between 1000
μmol L-1 and 1400 μmol L-1 have been reported
as thresholds that can be used for SCK (Duffield, 2000). A number of cowside
tests have been evaluated for the detection of ketone levels in serum,
milk or urine (Geishauser et al., 1997, 2000). Most of these tests
lack sensitivity as compared to serum BHB, which remains the gold standard
for studying ketosis.
Limited information is available regarding the prevalence of sub-clinical
ketosis in dairy herds in Fars province, Iran. The objectives of this
study were to determine the relationship between sub-clinical ketosis
and liver injuries within the first two months of lactation in high producing
Iranian Holstein cows. Results from this study would provide fundamental
knowledge for improving dairy production in Fars province, Iran. Serum
glucose, BHB and NEFA concentrations and absence of any signs of clinical
disease were considered as sub-clinical ketosis and serum AST, GGT, triglyceride,
VLDL-cholesterol and cholesterol concentrations were considered as liver
function parameters.
MATERIALS AND METHODS
A total of 77 Holstein cows within the first two months of lactation
(38 cows in the first month and 39 cows in the second month of lactation)
with high-producing records were randomly selected from three commercial
dairy herds that had a total of 530 cows in Fars province, Iran in year
2006. The animals were kept in free-stall housing.
All diets were based on alfalfa hay, corn silage and a combination of
concentrates including barley, corn, beet pulp, soyameal, wheat bran,
cotton seed meal, urea, fat powder and mineral and vitamin supplements.
Health and fertility records were maintained on all herds by the dairyman
and their veterinarians. Signs of clinical diseases, including clinical
ketosis, such as hard dry feces, diminished appetite, decreased milk production
and loss of body weight were noted.
For the analysis of serum biochemical parameters, blood samples were
collected from the coccygeal vein into plain vacutainers and the serum
was separated after centrifugation for 15 min at 750 x g at room temperature.
Any hemolyzed samples were discarded. Serum samples were stored at -20
°C until analyzed. Biochemical analysis including serum glucose was
carried out using the glucose oxidase method, BHB by the Williamson-Melanbaye
method (RANBUT Kit, RANDOX Com. UK), NEFA by the Matsubara method (NEFA
Kit, RANDOX Com. UK), AST by the modified method of Reitman-Frankel, GGT
by the modified method of Szasz, cholesterol by the modified method of
Abell-Kendall/Levey-Brodie (A-K) and triglyceride by the McGowan method
(Burtis and Ashwood, 1999). Serum VLDL-cholesterol was measured according
to Friedewald et al. (1972). A cutoff point of 1000 μmol L-1
serum BHB (Radostitis and Blood, 2000) was used to distinguish healthy
cows from cows with SCK.
The data in the first two months of lactation were analysed with independent
t-test. The correlations between different parameters were determined
with Spearman correlation test. All statistics were performed using SPSS
software for windows, version 6.0 (Norusis, 1993).
RESULTS
In the first month of postpartum period, serum concentration of glucose
in cows with SCK was significantly lower (p<0.05) than healthy cows
but the concentrations of BHB, NEFA, triglyceride and VLDL were higher
(p<0.05) in cows with SCK compared with healthy cows. In the second
month of lactation the serum concentration of BHB and NEFA were higher
(p<0.05) in cows with SCK compared with healthy cows (Table
1).
Within the first two months of lactation serum concentrations of BHB,
NEFA, triglyceride and VLDL were higher (p<0.05) in cows with SCK.
In the first month of lactation, the serum GGT concentration in healthy
cows was positively correlated with cholesterol (R = 0.533, p<0.05),
triglyceride and VLDL (R = 0.444, p<0.05) and in SCK cows, there was
a negative correlation (R = -0.576, p<0.05) between glucose and BHB
but a positive correlation between cholesterol and GGT (R = 0.761, p<0.05)
was noted. In the second month of lactation in healthy cows only a positive
correlation (R = 0.547, p<0.05) was observed between glucose and GGT.
Overall, in the first two month of lactation in healthy cows there was
a positive correlation (R = 0.409, p<0.05) between glucose and GGT
and in SCK cows positive correlations were observed between glucose and
cholesterol (r = 0.403, p<0.05) and GGT and cholesterol (R = 0.388,
p<0.05).
Table 1: |
Serum parameter concentration in the first, second
and the first two months of lactation periods (Mean ± SE) |
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1: Beta-hydroxybutyrate, 2: Aspartate aminotransferase,
3: Gamma-glutamyl transferase, 4: Triglyceride, 5: Very low density
lipoprotein and 6: Nonesterified fatty acid, *: Significant at p<0.05,
SE: Standard Error |
DISCUSSION
The results of this study showed that the concentration of serum BHB
in SCK cows within the first two months of lactation was significantly
higher than that of healthy cows, possibly due to higher energy demands
of cows at this stage which is in agreement with the results of Dann et
al. (2005), LeBlanc et al. (2005) and Padilla et al.
(2005). Ketosis is a disease related to the high rate of glucose utilization
in the mammary gland and the inability of the cows to meet this glucose
demand by normal physiology. On the other hand, SCK is defined as elevated
concentrations of circulating ketone bodies (due to mobilization of NEFA)
in the absence of clinical signs of ketosis (Dann et al., 2005).
These compounds are important source of energy when carbohydrate levels
are reduced (Duffield, 2000). SCK is important because it may remain undetected
and yet have effects on productivity which parallel those elicited by
clinical ketosis. Prevalence of SCK increases from primiparous to multiparous
cows (Detilleux et al., 1994) and also other factors such as age
(Andersson, 1988), season (Whitakar et al., 1993) and breed (Andersson,
1988) can affect its prevalence. SCK may start at serum BHB concentrations
above 1000 μmol L-1 and clinical ketosis at about 2600
μmol L-1. However, at exactly what level individual cow
will show clinical sign is extremely variable (Andersson, 1984). Also
it has been reported that a range of blood BHB concentrations from 1000
to 1400 μmol L-1 can be used for detecting SCK (Whitakar
et al., 1993). At all sampling times, the serum BHB concentrations
in SCK cow were significantly (p<0.05) higher than 1000 μmol L-1
(but less than 2600 μmol L-1) which is considered as SCK
condition (LeBlanc et al., 2005; Walsh et al., 2007b). BHB
is synthesized from absorbed butyrate in the rumen epithelium and by the
ketogenesis of hepatocytes in the conversion of long chain fatty acids
during fat mobilization. In SCK cows, BHB is the predominant circulating
ketone body and is relatively stable in whole body, plasma or serum (Dohoo
and Martin, 1984). Blood glucose and ketone bodies can be used as a measure
of energy status of the animal. There is relatively weaker degree of homeostatic
regulation of BHB than glucose which means that BHB concentrations are
less constrained physiologically and more likely is a reflection of nutritional
status than blood glucose (Herdt, 2000).
The significant negative correlation between BHB and glucose concentration
(Padilla et al., 2005) in the first month of lactation is in the
line of the fact that hypoglycemia is the driving force in bovine sub-clinical
and clinical ketosis, which ends to ketonemia (Bruss, 1997). It has been
shown that in SCK, cows can become ketonemic without the presence of significant
hypoglycemia (Grohn et al., 1983), as was seen in the second and
first two months of lactation in the present study.
The significantly (p<0.05) higher triglyceride concentration in SCK
cows in present study is in agreement with the findings of Holtenius and
Hjort (1990) and Drackley et al. (1992). The higher serum triglyceride
concentration in SCK cows in this study is not in agreement with other
studies (Reichel and Sokoi, 1987) with fatty liver syndrome cows and liver
injuries. This shows the normal function of liver in this study in SCK
cows. The higher liver lipoprotein synthesis will decrease the incidence
of fatty liver syndrome and liver injuries (Grummer, 1995). The lower
serum glucose and higher serum BHB concentrations in cows with SCK in
the first month of lactation in the present study is in agreement with
the findings of Dann et al. (2005), Radostits and Blood (2000)
and Padilla et al. (2005). Regarding the circulating glucose, there
is conflicting data in the literature. Bremmer et al. (2000) reported
that glucose concentration is decreased in response to energy restriction
in the diet, while Canfield and Butler (1991) concluded that there is
little influence of the energetic status of the animal on the blood glucose
concentration. Overall blood glucose is an insensitive measure because
it is subjected to tight homeostatic regulation. The same trend for BHB
was observed for cows with SCK in the first, second and within two months
of lactation period.
Circulating levels of non-esterified fatty acids (NEFA) and BHB are valid
measurements of energy metabolism (Dann et al., 2005). Serum NEFA
greater than 0.4 mmol L-1 has been proposed to identify the
negative energy balance and SCK (Stokol and Nydam, 2005). In the present
study, the concentration of serum NEFA in SCK cows at all stages of sampling
were higher than 0.4 mmol L-1. The SCK cows mobilized adipose
lipid reserves to support the negative energy balance and had elevated
concentrations of BHB and NEFA in serum and had lower concentrations of
glucose (Dann et al., 2005; Padilla et al., 2005). Since
the serum concentration of VLDL is correlated with serum triglyceride
(Friedewald et al., 1972) both can be used as indicators of energy
status of the cow. A decrease in serum triglyceride has been reported
in liver injuries and fatty liver syndrome conditions (Reichel and Sokoi,
1987), which is due to low capacity of liver lipoprotein synthesis (Grummer,
1995). In the present study, the serum triglyceride and VLDL concentrations
were higher (p<0.05) in SCK cows compared with healthy cows which is
another sign of the absence of liver injuries in the former cows. Concentrations
of triacylglycerol usually increase in parallel with those of total lipid
(Grum et al., 1996). There was no significant differences between
healthy and SCK cows for serum AST, GGT and cholesterol concentrations
at all sampling times. Liver injuries is associated with higher serum
hepatic enzymes e.g., AST and GGT (Smith, 1996).The serum concentration
of GGT increases in liver and bile duct malfunctions (Steen et al.,
1997) and liver is the main source of serum GGT (Kaneko, 1989), while
serum concentration of AST increases due to fat accumulation in the liver
which results in high hepatocytes membrane permeability and is a good
tool for detection of early metabolic liver diseases (Karsai and Schafer,
1984). In the present study, fatty infiltration did not cause liver damage
as indicated by liver-specific enzymes (AST and GGT) measured in serum
at all sampling times which is in the line of the findings of Dann et
al. (2005). Steen et al. (1997) reported that AST activity
was greater in cows with ketosis and hepatic lipidosis than in cows that
were healthy.
In conclusion, the results of this study show that the prevalence of
SCK in Fars province is considerable and measuring serum BHB as a routine
monitoring program could be beneficial for dairy herds. In order to prevent
the economic loss due to SCK, early treatment of SCK cows is important
and prevention of the disease has to be achieved through good nutritional
programs in the dry and early lactation periods.
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