Research Project Veterinary Medicine University Utrecht J. Hoekstra 3259145 8 April 2011 Project tutors: dr. T. Rukkwamsuk & dr. J.T. Schonewille

Table of contents
 Abstract  Introduction  Materials and methods  Results & discussion  Conclusion  Acknowledgements  References  Attachments: questionnaires page 3 page 4 - 8 page 9 page 10 – 23 page 24 page 25 page 26 – 28 page 29 – 54

Abstract
Negative energy balance was studied in 26 dairy cows from small-holder farms in Thailand. Blood samples were collected at 4 weeks prepartum and 0 to 3 days postpartum for evaluation of negative energy balance and to study the relationship between concentrations of serum non-esterified fatty acids, concentrations of serum glucose and body condition score. Body condition score was determined 4 weeks before calving, 1 week before to 3 days after calving and 4 weeks after calving. The results revealed that the concentration of non-esterified fatty acids was significantly higher after parturition compared with the concentration before parturition. The concentrations of serum glucose and body condition score were significantly decreased after parturition in comparison with before parturition. In conclusion, periparturient dairy cows of small-holder farms in Thailand had some degrees of negative energy balance as observed by increased serum non-esterified fatty acids concentration, decreased serum glucose concentration and decreased body condition score.

Introduction
In Thailand, most dairy cows are kept in small-holder farms with an average of forty cows, in which the farmers are confronting several production diseases particularly related to poor feed and feeding management. An important period is the periparturient period or transition period, from 3 weeks before to 3 weeks after calving. In this period, dairy cows are predisposed to poor health condition. Most health problems occur during this period (Drackley, 1999; Goff et al., 1997; Mulligan et al., 2006). The periparturient period is characterised by adaptations to the demands of lactation. The metabolic state of the cow changes from a non-lactating state to a lactating state. Thirty to fifty percent of the dairy cows are affected by some forms of metabolic or infectious diseases in the periparturient period, such as ketosis, metritis, milk fever and mastitis (Drackley, 1999; Goff et al., 1997; Le Blanc, 2010). Negative energy balance The change to a lactating state causes a change in energy demand. Near the end of gestation, the daily development of the fetus requires 3.43 MJ of energy (Goff et al., 1997). The metabolizable energy (ME) requirement for maintenance is 44.2 MJ for a 500 kg cow (Moe et al., 1972). Thus the development of the fetus requires 7.8% of the ME. The onset of lactation increases the total energy requirements by approximately a fourfold, compared with the energy requirement before parturition (Block et al., 2001). The production of 10 kg colostrum requires 45.98 MJ of energy (Goff et al., 1997). Generally, the energy intake in this period is not sufficient to meet this increased energy demand, because the cow cannot obtain that amount of energy from the diet (Block et al., 2001; Collard et al., 2000; Goff et al., 1997; Reist et al., 2002). The energy intake is thus lower than the energy needed for the high milk production. This results in negative energy balance (NEB), which begins a few days before calving and reaches its lowest level about two weeks postpartum (Kim et al., 2003). Essentially all cows develop some degree of NEB in the periparturient period (Ametaj, 2005; Le Blanc, 2010; Reist et al., 2002). During the period of NEB, stored body fat will be mobilized in the form of non-esterified fatty acids (NEFAs) and protein in the form of amino acids. The release of NEFAs is stimulated by hormone-sensitive lipase (Melendez et al., 2009). The adipose mobilisation leads to high blood serum concentrations of NEFAs. NEFAs are partially carried in the blood to the liver, where they are esterified to triacylglyceride (TAG). The TAG can be transported as very-low-density-lipids (VLDL), which go to the mammary gland for milk fat production and to other tissues to serve as an energy source. The capacity to export TAG in the form of VLDL is limited. If the TAG cannot be excreted as VLDL, it is stored in the liver. NEFAs can also be used for the production of ketone bodies (Roche et al., 2009). Figure 1 shows the lipid metabolism in adipose tissue, liver and mammary gland (adapted from Drackley, 1999). If energy obtained from the diet and the amount of mobilised fat energy meet the energy requirement, milk production is maintained and the cows remain healthy. On the other hand, when the energy demand is higher than the available energy, the NEB is profound and the cow will develop health problems (Drackley, 1999; Šamanc et al., 2010). The severity of the NEB and the time it takes to recover is critical for the health status and productivity of the cow (Reist et al., 2002).

Figure 1: relationship between lipid metabolism in adipose tissue, liver and mammary gland. Adapted from Drackley (1999) Consequences of NEB NEB is associated with metabolic diseases, including ketosis, fatty liver and displaced abomasum (Collard et al., 2000) Cows with a greater degree of NEB at calving are more likely to have a retained placenta or postpartum uterine infections (Le Blanc 2010; Melendez et al., 2009). High NEFA concentrations approximately one week before expected calving (> 0.4 mmol/L) are associated with an increased risk of left displaced abomasum, retained placenta, ketosis, mastitis and culling (Le Blanc, 2010). Cows with a NEFA concentration ≥ 1.2 mmol/L at calving have an increased risk of clinical mastitis and milk fever compared with a NEFA concentration < 1.2 mmol/L (Melendez et al., 2009). According to Collard et al. (2000), mobility problems such as laminitis are phenotypically associated with NEB. But it is difficult to determine whether NEB is the cause or the result of mobility problems. Cows with mobility problems spend more time laying down, which result in a lower feed intake which may lead to NEB. NEB is associated with relatively low milk production. Health problems during the periparturient period lead to a decrease in milk yield, not only during the time of disease but often during the entire lactation period (Drackley, 1999). Indeed, in a study conducted with Thai dairy cows, cows suffering from NEB did not produce optimum milk yields (Rukkwamsuk, 2010). Furthermore, NEB is associated with reproductive problems. Yawongsa et al. (2003) found suboptimal reproductive performances in Thai cross-bred Hollstein Friesian cows. The reproductive problems might be a consequence of periparturient NEB. In a study conducted with Thai dairy cows, cows suffering from NEB had a delayed first estrus after calving (Rukkwamsuk 2010). Collard et al. (2000) also concluded that NEB extends the postpartum interval to first ovulation. Fatty liver and ketosis The most common health problems associated with NEB are fatty liver and ketosis (Šamanc et al., 2010).

Fatty liver is characterized by the accumulation of fat in the liver. NEB causes the mobilisation of large quantities of fatty acids. If mobilisation and accumulation of fatty acids from adipose tissue is greater than the capacity of the liver to secrete or burn the fatty acids, the fatty acids will be stored as triacylglycerides (TAG) in the liver. If the accumulation of lipid in the liver is severe, it may result in fatty liver (Ametaj, 2005; Drackley, 1999; Goff et al., 1997; Overton 2001; Šamanc et al. 2010). Ketosis is also caused by the mobilisation of fatty acids. Ketone bodies are produced from NEFAs in the liver. Ketosis is an important process in early lactation. Vital organs such as the brain cannot use fatty acids as an energy source. The majority of the available glucose is used by the mammary gland. Thus, ketone bodies are needed to survive the early lactation period (Roche et al., 2009). Hyperketonemia develops if the production of ketone bodies is higher than the oxidation (Risco, 2010). Clinical ketosis becomes clinically evident from 10 days to 3 weeks after parturition (Goff et al., 1997). Cows with clinical ketosis are lethargic, have a reduced milk production and reduced feed intake (Šamanc et al., 2010; Merck Veterinary Manual, 2008). Risk factors associated with NEB
Body condition score
Body condition score (BCS) is an indicator of the energy balance of the cow (Šamanc et al., 2010). During NEB, the BCS decreases because NEB leads to the mobilisation of body reserves. The lowest BCS is reached one to two months after calving (Collard et al., 2000; Drackley, 1999; Šamanc et al., 2010). The recommended BCS at calving lies between 3.0 and 3.25 (Mulligan et al., 2006; Overtone, 2001; Roche et al., 2009). Overfeeding during the dry period predisposes dairy cows to the development of fatty liver postpartum (Top, A.M. van den et al., 1996, Rukkwamsuk, T. et al., 1998). It is generally assumed that over-conditioned cows at calving have a greater decrease in feed intake in comparison with cows with a lower BCS (Grummer et al., 2004, Mulligan et al., 2006; Murondoti et al., 2004, Roche et al., 2009; Šamanc et al., 2010, Janovick et al., 2011). The over-conditioned cows therefore will have a more severe NEB, because their energy intake is lower. The mobilisation of fat in the early lactation period will be higher, which results in a higher risk of developing fatty liver (Mulligan et al., 2006; Roche et al., 2009; Šamanc et al., 2010). Research has shown that cows that are overfed during the dry period have a greater incidence of fatty liver. (Drackley, 1999). However, other research has shown that the dry matter intake of over-conditioned cows is only slightly lower than de dry matter intake of the control group (Van den Top et al., 1996). The overfed cows did have a higher plasma NEFA concentration postpartum, indicating that they had a higher rate of lipolysis compared with the control group. This indicates that over conditioned cows had a greater NEB postpartum compared with control cows, despite that the dry matter intake did not differ. Lipolysis in overfed cows may be suppressed before parturition. As a result, the livers of overfed cows are less adapted for the metabolization of NEFAs around calving. After calving, overfed cows have a higher NEFA concentration and less adapted livers in comparison with cows that are in normal condition, leading to the accumulation of greater amounts of TAG in the liver (Van den Top et al., 1996, Rukkwamsuk et al. 1998). Other research has shown that the oxidation of NEFAs in the liver after calving is reduced in overfed cows (Murondoti et al., 2004). This may also contribute to the development of fatty liver.

Furthermore, cows with a calving BCS of > 3.5 have a higher risk of ketosis, compared with cows with a calving BCS of 3.25 (Gillund et al., 2001; Roche et al., 2009; Šamanc et al., 2010). A higher loss of BCS postpartum is associated with higher fat mobilisation and thus a higher risk of developing fatty liver (Šamanc et al., 2010). A higher loss of BCS is also associated with a higher risk of ketosis, abomasal displacement and milk fever (Kim et al., 2003; Gillund et al., 2001).
Lactation number
There is possibly an association between the lactation number and the energy status. This relationship is studied by Wathes et al. (2007). They found that primiparous cows had higher NEFA concentrations at calving in comparison with multiparous cows. One week before calving, the NEFA concentration was already elevated. Thus, primiparous cows were in worse energy status before calving in comparison with multiparous cows. After calving, the concentration of NEFAs did not increase further and started to decrease two weeks postpartum. Multiparous cows on the other hand, had the peak NEFA concentration at three weeks postpartum (Wathes et al., 2007). The prevalence of ketosis is higher with increasing lactation number. Multiparous cows thus suffer from a greater NEB postpartum than primiparous cows. The increase in NEFA concentration occurs later than in primiparous cows.
Milk fever
Another important metabolic disease is milk fever. Milk fever, or hypocalcemia, is defined as a blood calcium concentration lower than 2.0 mmol/L. It occurs within 72 hours after calving and is caused, just as NEB, by the onset of lactation (Merck Veterinary Manual, 2008). Milk fever and NEB are both important periparturient disorders. These two health problems may be linked through the feed intake and there may be an association between these two problems. Milk fever leads to a greater decline in feed intake after calving. This increases the severity of NEB, the mobilisation of fat and the risk of fatty liver and ketosis (Goff et al., 1997; Melendez, 2009). However, other researchers did not find an association between NEB and milk fever: they found that elevated NEFA concentrations one week before calving are not associated with greater incidences of milk fever (Drackley, 1999). And according to Roche et al. (2009), cows in NEB prepartum remain periparturient eucalcemia more effectively than cows who are not in NEB. Thus the question remains if there is a relationship between NEB and milk fever.
Heat stress
Heat stress is an important factor contributing to infertility of lactating dairy cows in a tropical climate (Rukkwamsuk et al., 2004). However, research conducted with Thai dairy cows kept under cooled conditions in commercial dairy herds showed that 68.4% of these cows had periparturient fatty liver (Rukkwamsuk et al., 2004). There are no data about the frequency of fatty liver in Thai dairy cows kept under normal Thai conditions. Diagnostic tools NEB is associated with lowered blood glucose concentrations and increased non-esterified fatty acids (NEFA) in the blood (Rukkwamsuk et al., 2006).

According to Reist et al. (2002), concentrations of NEFA correlated stronger with the energy balance than other blood metabolites, including glucose and the ketone body β hydroxybutyrate. They concluded that NEFA is one of the most informative traits for the estimation of energy balance (Reist et al., 2002). According to Le Blanc, serum NEFA concentrations measured 4 to 10 days before expected calving provide a useful component of assessment of peripartum health (Le Blanc, 2010). The concentration of NEFAs begins to rise 2 to 4 days prepartum and its peak is about 3 days postpartum. The increase starts earlier and is greater in cows that will experience metabolic disease (Le Blanc, 2010). The NEFA concentration 4 weeks before expected calving could be served as a control value, because at that moment, cows are raised on their maintenance requirement. During parturition, cows are forced to become NEB, causing a rise in the serum NEFA concentration. The change in NEFA concentration 4 weeks before calving and 0 to 3 days after calving is an indicator for the severity of NEB: a higher rise in serum NEFA concentration means a more severe NEB. To avoid postprandial variation in the serum NEFA concentration, samples should be collected around the same time (Le Blanc, 2010). The glucose concentration is another indicator of the energy balance. NEB results in a decrease of the serum glucose concentration. The decrease in serum glucose concentration leads to an increased lipolysis of adipose tissue, which leads to an increase in serum NEFA concentrations (Rukkwamsuk 2010). As stated earlier, the BCS is also an indicator of the energy balance. Over-conditioning at calving is associated with a more severe NEB and higher BCS loss is associated with fatty liver, ketosis and more severe NEB. The BCS and the BCS loss may be a useful method to determine the severity of NEB. Aim of the study Most Thai dairy cows are raised in small-holder farms, which are not well-managed regarding to nutrition and herd health management (Rukkwamsuk, 2010). For managing herd health, it is important to know the risk factors for NEB and to prevent the development of NEB. The risk factors for NEB are known under temperate conditions. The most critical risk factor under temperate conditions is the dry matter intake. The aim of the current study is to determine the prevalence of negative energy balance in Thai dairy cows and trace out the most relevant risk factors of NEB under the typical Thai conditions. The relationship between serum glucose concentration, serum NEFA concentration and BCS will be determined. It is expected that cows with a higher BCS at calving will have a more severe NEB and thus lower serum glucose concentrations and higher serum NEFA concentrations. This study will also determine if there is an association between NEB and milk fever in Thai dairy cows. It is anticipated that the outcome of this study provides a scientific basis for further optimalization of rations for Thai dairy cows.

Materials and methods
Animals and sampling 26 pregnant cross-bred Holstein-Friesian cows were randomly selected from the Dairy Cooperatives in Western Part of Thailand according to their calving date during January to March 2011. Questionnaires are used to collect feed and feeding management as well as pertinent data of cows and herds. Body condition scores (BCS) from all cows are recorded 4 weeks before expected calving, 1 week before to 3 days after calving (which will be referred to as week 0) and 4 weeks after parturition. The BCS is determined by palpation and visual examination, using a five-point scale with quarter-part divisions (1 = emaciated to 5 = obese). Blood samples are collected from all cows at 4 weeks before expected calving date and at 0 to 3 days after parturition (which will be referred to as week 0) to determine serum concentrations of glucose and NEFA. Blood samples are taken from the vena coccygea. About 6 ml blood is collected in a Vacuette® serum tube. Blood and serum are separated with centrifugation and the serum is aspirated, transferred into a plastic transport tube and stored by -10 degrees Celsius until analyzed. Data on milk production, milk composition and feed intake will also be collected. Sample analyses Serum concentrations of glucose and nonesterified fatty acid are determined using spectrophotometry with commercially available test kits. Statistical analysis Prevalence data are reported as descriptive statistics. Comparison of blood parameters and body condition scores between pre- and postparturition is analyzed using repeated measures ANOVA. Comparison of data between groups are analyzed using the paired Student t test. Relationship between different variables is determined with the Spearman’s rank correlation coefficient. The two-sided level of statistical significance is preset at P ≤ 0.05.

Results and discussion
Unless mentioned otherwise, 26 cows are included in the analysis. Body condition score The mean (± standard deviation (SD)) BCS 4 weeks before parturition was 3.10 ± 0.45; the mean BCS at week 0 was 3.11 ± 0.61; and the mean BCS 4 weeks after parturition was 2.72 ± 0.33 (figure 2). The mean BCS found in this research are similar with results found in two previous studies conducted with Thai dairy cows raised in small-holder farms. The average BCS at week 0 was 3.08 ± 0.08 (Rukkwamsuk et al. 2006) and 3.02 ± 0.64 (Rukkwamsuk, 2010). The average BCS 4 weeks after calving was 2.48 ± 0.04 (Rukkwamsuk et al. 2006). The BCS 4 weeks before calving is lower than the BCS of 3.79 ± 0.55 found in a Western study (Samanc et al. 2010). The BCS at calving in the Thai studies are also lower compared with the mean BCS at calving of 3.60 ± 0.34 in another Western study (Melendez et al. 2009). The findings of the Thai studies indicate that Thai dairy cows have lower BCS in comparison with Western countries. The recommended BCS at calving lies between 3.0 and 3.25 (Mulligan et al., 2006; Overtone, 2001; Roche et al., 2009). This indicates that on average, Thai dairy cows have the recommended BCS at calving. Most dairy cows lost body condition during the first month of lactation: the BCS 4 weeks after parturition was significantly lower than the BCS 4 weeks before parturition (P < 0.001) and the BCS at week 0 (P < 0.001). This suggests that the Thai dairy cows in this study suffered from NEB after calving (Rukkwamsuk et al. 2006, Rukkwamsuk 2010). This in agreement with the notion that essentially all cows develop some degree of NEB in the postparturient period (Ametaj, 2005; Le Blanc, 2010; Reist et al., 2002). It is expected that the mean degree of NEB is modest, because the mean loss of BCS is modest. 4 3,5 3 2,5 2 1,5 1 0,5 0 -4 0 4
Weeks from parturition
Figure 2: mean body condition score of 26 cows. The mean BCS at -4 weeks from parturition was 3.10 ± 0.45, the mean BCS at week 0 was 3.11 ± 0.61, and the mean BCS 4 weeks after parturition was 2.72 ± 0.33. Data represent means and SD as error bars.

Blood biochemistry Serum glucose and serum NEFA concentrations are shown in figures 3 and 4 respectively.
Glucose
The mean serum glucose concentration 4 weeks before parturition was 54.31 ± 10.67 mg/dL. The mean serum glucose concentration at week 0 was 42.25 ± 10.17 mg/dL. The serum glucose concentration after calving was significantly lower than the serum glucose concentration before calving (P < 0.001), which suggests that the cows suffered from NEB immediately after calving. The observed serum glucose concentrations in the current study were higher than the concentrations found in a Western study: 41 ± 1.3 mg/dL one month before parturition and 39 ± 1.7 mg/dL at calving (Kim et al., 2003).
NEFA
The mean serum NEFA concentration 4 weeks before parturition was 0.38 ± 0.20 mmol/L. This is in comparison with the normal value of 0.24 ± 0.07 mmol/L that was reported earlier in Thailand (Rukkwamsuk et al. 2006). The mean serum NEFA concentration at week 0 was 0.72 ± 0.27 mmol/L. This is higher than the mean serum NEFA concentration of 0.58 ± 0.05 mmol/L 2 weeks after calving found in a previous study (Rukkwamsuk et al. 2006). However, it is difficult to compare the concentrations of week 0 with concentrations found 2 weeks postpartum, because the NEFA concentration will decrease to normal values in the first month after calving. The mean serum NEFA concentration after calving found in this study is lower than the mean serum NEFA concentration of 0.90 ± 0.47 mmol/L found in a Western study (Melendez et al. 2009). Melendez et al. (2009) found no association between a NEFA concentration of 0.90 mmol/L and the incidence of periparturient diseases. 30 20 10 0 70 60 50 40 -4 0
Weeks from parturition
Figure 3: serum glucose concentrations of dairy cows (n = 26) at -4 and 0 weeks from parturition. The mean glucose concentration -4 weeks from parturition was 54.31 ± 10.67 mg/dL. The mean glucose concentration 0 weeks from parturition was 42.25 ± 10.17 mg/dL, which was significantly lower than the concentration -4 weeks from parturition. Data represent means and SD as error bars.

1,2 1 0,8 0,6 0,4 0,2 0 -4 0
Weeks from parturition
Figure 4: serum NEFA concentrations of dairy cows (n = 26) at -4 and 0 weeks from parturition. The mean serum NEFA concentration at -4 weeks from parturition was 0.38 ± 0.20 mmol/L. The mean concentration at week 0 was 0.24 ± 0.07 mmol/L. Data represent means and SD as error bars. Van den Top et al. (1996) conducted a study in which dairy cows either had free access to food during the dry period (the test group) or were fed according to their energy requirements (the control group). Cows in the test group developed fatty liver postpartum, where cows in the control group did not. The mean serum NEFA concentration at parturition in the control group was 0.75 mmol/L, the mean serum NEFA concentration in the test group was 1.50 mmol/L (Van den Top et al., 1996). The mean serum NEFA concentration in the present study is in comparison with the mean serum NEFA concentration in the control group. This leads to the the conclusion that the NEFA concentrations found in the present study are unlikely to cause adverse health effects. However, other studies report a NEFA threshold of 0.60 mmol/L 3 to 14 days postpartum. Cows with a NEFA concentration above this threshold are at higher risk for periparturient diseases (Ospina et al. 2010a, Ospina et al. 2010b). The average serum NEFA concentrations in the present study were higher than these threshold, but none of the cows suffered from periparturient diseases. Another study found that cows with NEFA concentrations ≥ 1.2 mmol/L after calving had a higher risk of periparturient diseases (Melendez et al 2009). The serum NEFA concentration after calving was significantly higher than the serum NEFA concentration before calving (P < 0.001). The increased NEFA concentration is a result of the increase of adipose tissue lipolysis, caused by the decreased glucose concentration. This result suggests that the cows suffered from some degree of NEB immediately after calving. In earlier research conducted with Thai dairy cows raised in small-holder farms, the serum NEFA concentrations did not differ between before and after calving (Rukkwamsuk et al. 2006). However, in a more recent study conducted with Thai dairy cows, the NEFA concentration was significantly higher after parturition than before parturition (Rukkwamsuk, 2010). It is expected that the serum NEFA concentration rises after parturition. The concentration of NEFAs begin to rise 2 to 4 days before parturition and the highest concentration will be reached 3 days postpartum (LeBlanc, 2010). The rise of NEFA concentrations found in this study is thus according to the expectations.

Body condition score and serum NEFA concentration Cows in this study suffered from some degree of NEB within 3 days after calving. During NEB, the BCS decreases because NEB leads to the mobilisation of body reserves. The lowest BCS is reached one to two months after calving (Collard et al., 2000; Drackley, 1999; Šamanc et al., 2010). It is thought that changes of BCS during the periparturient period could be an indirect parameter for determination of the severity of NEB in dairy cows (Rukkwamsuk, 2010). However, there was no correlation between the change of BCS between 4 weeks before and 4 weeks after calving and the change in serum NEFA concentration (r = -0.021). This indicates that the changes of BCS during the periparturient period cannot be used to determine the severity of NEB in Thai dairy cows. There was a negative, significant correlation between the change of BCS between 4 weeks before calving and week 0 and the change of serum NEFA concentration (correlation coefficient: -0.490; correlation is significant at 0.001 level (2-tailed)). This indicates that cows with a higher loss of BCS before calving have a lower increase of serum NEFA concentration. This is not what is expected, because a higher loss of BCS is associated with a higher risk of ketosis, abomasal displacement and milk fever and thus with a more severe NEB (Kim et al., 2003; Gillund et al., 2001). A positive correlation would be expected. A possible explanation for the finding in the present study, is that only 7 out of 26 cows lost BCS between 4 weeks before calving and week 0 and there was no significant difference between the BCS 4 weeks before calving and week 0. This might indicate that the Spearman’s rank correlation cannot be used in this case. Therefore, cows were divided in three groups: cows who lost BCS between 4 weeks before calving and week 0 (n = 7; mean loss was 0.36 ± 0.20) and cows who gained BCS (n = 8; mean gain was 0.41 ± 0.19) and cows who did not lose or gain BCS (n = 11) during that period. The mean change in serum NEFA concentrations were 0.47 ± 0.23, 0.17 ± 0.16 and 0.38 ± 0.39 mmol/L respectively (figure 5). The means were compared with a paired samples T-test and a significant difference was found between cows who gained BCS and cows who lost BCS (P = 0.025). Figure 5: the change in serum NEFA concentration for cows who lost BCS, gained BCS or neither gained nor lost BCS between 4 and 0 weeks before parturition. Data represent means and SD as error bars.

Thus cows who lost BCS during the last month before calving had higher serum NEFA concentrations than cows who gained BCS in that period. In the current study, change of BCS in the last month of gestation was an indirect parameter for the severity of NEB. The mean change in serum NEFA concentration was not significantly different between cows with no change of BCS and the other two groups. However, the daily feed intake is also an important determinant of the NEFA value. A change in NEFA based on two values measured at 4 weeks and 0 weeks from parturition might not be very meaningfull. Cows with a calving BCS of > 3.5 have a higher risk of ketosis, compared with cows with a calving BCS of 3.25 (Gillund et al., 2001; Roche et al., 2009; Šamanc et al., 2010). There was no correlation between the BCS at calving and the change in serum NEFA concentration (r = -0.131) in this study. This might be due because most cows included in this study had a BCS lower than the optimal BCS at calving. Therefore, cows were divided in three groups: cows with a BCS of < 3.0 (n = 13), cows with a BCS between 3.0 and 3.25 (n = 4) and cows with a BCS of > 3.25 (n = 9) at calving. The mean changes in serum NEFA concentrations were 0.38 ± 0.29, 0.45 ± 0.52 and 0.22 ± 0.21 mmol/L respectively (figure 6). It is expected that over-conditioned cows lose more BCS in the first month after calving, because they suffer from a more severe NEB. Cows with a BCS > 3.25 had a mean BCS loss of 0.92 ± 0.28, while cows with a BCS between 3.0 and 3.25 lost 0.31 ± 0.24 BCS and cows with a BCS < 3.0 lost 0.04 ± 0.22 BCS in this first month of lactation. Cows with a BCS > 3.25 lost significantly more BCS than cows with a BCS < 3.0 (P = <0.001). This indicates that over-conditioned cows in this study lost more BCS after calving and were thus in a more severe NEB than cows with a lower BCS. This is according to the expectations. It is not in agreement with the change of the serum NEFA concentration, which is lower in cows with a BCS > 3.25. A higher loss in BCS indicates that there is more fat mobilisation, which would lead to a higher change in serum NEFA concentration. However, cows in this study were not over-conditioned. Even in the group with BCS > 3.25, the mean BCS was 3.83 ± 0.28. This is higher than recommended, but some authors recommended a BCS between 3.25 to 3.5 (Overton, 2001). The mean BCS in the three groups lie close together, which may be a possible explanation that the result differ from the results in other studies. There is also no information about the feed intake. The cows with the optimal BCS (between 3.0 and 3.25) did not lose significantly less BCS than cows with a BCS > 3.25 (P = 0.092). Cows with a BCS < 3.0 lost significantly less BCS than the cows with the optimal BCS (P = 0.015). This might indicate that the cows with an optimal BCS were in worse NEB, but they did not have a significant higher change in NEFA concentration. However, there were only 4 cows with an optimal BCS and the serum NEFA concentration is measured after calving, before its peak 3 days postpartum.

Figure 6: the change in serum NEFA concentration for cows with a BCS < 3.0 (mean change: 0.38 ± 0.29), a BCS between 3.0 and 3.25 (mean change: 0.45 ± 0.52) and a BCS > 3.25 (mean change: 0.22 ± 0.21). Data represent means and SD as error bars. Figure 7: The mean BCS for cows with a BCS < 3.0 was 2.60 ± 0.22 at 0 weeks and 2.56 ± 0.23 at 4 weeks from parturition. The mean BCS for cows with a BCS 3.0 – 3.25 was 3.13 ± 0.14 at 0 weeks and 2.81 ± 0.13 at 4 weeks from parturition. The mean BCS for cows with a BCS > 3.25 was 3.83 ± 0.28 at 0 weeks and 2.92 ± 0.40 at 4 weeks from parturition. Data represent means and SD as error bars.

Figure 8: the serum NEFA concentration -4 and 0 weeks from parturition for cows with a BCS < 3, between 3 and 3.25 and > 3.25. The serum NEFA concentration at -4 weeks was 0.35 ± 0.20, 0.36 ± 0.21 and 0.44 ± 0.20 respectively. The serum NEFA concentration at 0 weeks was 0.74 ± 0.28, 0.81 ± 0.41 and 0.66 ± 0.20 respectively. Data represent means and SD as error bars. Concluding, the result in this study are not according to the expectations and result found in previous studies. There was no correlation between the change of BCS between -4 and +4 weeks from parturition and the change in serum NEFA concentration. BCS loss one month before calving was associated with a higher change in serum NEFA concentration between 4 and 0 weeks from parturition and are therefore at higher risk to develop more severe NEB. Body condition score and serum glucose concentration There was no significant correlation between the glucose concentration at week 0 and the BCS at week 0 (r = 0.211). This is not in agreement with previous research, which stated that over-conditioned cows will suffer from more severe NEB and thus have lower serum glucose concentrations. A negative correlation between the BCS at week 0 and the glucose concentration at week 0 was expected. Cows were divided in three groups: cows with a BCS of < 3.0 (n = 13), cows with a BCS between 3.0 and 3.25 (n = 4) and cows with a BCS of > 3.25 (n = 9) at calving. The mean serum glucose concentrations 4 weeks before parturition were 49.11 ± 10.35, 58.48 ± 12.62 and 59.97 ± 8.89 respectively; the serum glucose concentrations after calving were 40.84 ± 8.86, 45.48 ± 12.62 and 44.29 ± 7.28 respectively (table 1 en figure 9). There was no significant difference in the serum glucose concentration after calving. As stated above, it was expected that cows with a higher BCS had lower serum glucose concentrations after calving. The serum glucose concentrations of cows with a BCS > 3.25 are higher, although this is not significant. However, as mentioned earlier in this research, the average BCS of the cows in the group > 3.25 was only slightly higher than recommended. It might not be justified to draw conclusions about over-conditioning and NEB, because the cows in this study were not really over-conditioned.

Table 1
BCS < 3.0 BCS 3.0 – 3.25 BCS > 3.25 Serum glucose -4 weeks (mg/dL)
49.11 ± 10.35 58.48 ± 12.62 59.97 ± 8.89
Serum glucose 0 weeks
40.84 ± 8.86 45.48 ± 12.62 44.29 ± 7.28 Figure 9: data represent means and SD as error bars. Cows were divided in two groups: cows with hypoglycemia (serum glucose concentrations < 45 mg/dL) at week 0 (n = 15) and cows without hypoglycemia at week 0 (n = 11). Tabel 2: group 1 are cows with hypoglycemia after calving.
Group 1 BCS -4 weeks
2.98 ± 0.48
BCS 0 weeks
3.05 ± 0.65
BCS +4 weeks
2.73 ± 0.42
Group 2
3.25 ± 0.37 3.18 ± 0.58 2.70 ± 0.15
Group 1 ∆BCS -4 – 0 weeks
+0.067 ± 0.31
∆BCS 0 – 4 weeks
-0.32 ± 0.44
∆BCS -4 – 4 weeks*
-0.25 ± 0.38
Group 2
-0.022 ± 0.36 -0.48 ± 0.52 -0.55 ± 0.33 * Cows in group 2 lost significantly more BCS than cows in group 2 (P = 0.046)
Group 1 ∆NEFA -4 – 0 weeks (mmol/L)
0.34 ± 0.26
Glucose -4 weeks (mg/dL)
51.16 ± 12.16
Glucose 0 weeks (mg/dL)*
37.15 ± 6.78
Group 2
0.33 ± 0.38 58.62 ± 6.52 50.38 ± 4.35 * The glucose concentration in group 1 was significantly lower (P < 0.001) There was no significant difference between the BCS in the two groups. This is in agreement with the results of the Spearman’s rank correlation, where we did not find a correlation between the BCS at calving and the serum glucose concentration after calving.

There was a significant difference between the change in BCS between 4 weeks before and 4 weeks after calving and the glucose concentration after calving (P = 0.046). Cows with hypoglycemia immediately after calving lost significantly less BCS in this period than cows without hypoglycemia. This is not in agreement with the expectations, because it is expected that cows with a higher loss of BCS suffer from more severe NEB. It was expected that cows with hypoglycemia would have a significant higher change in serum NEFA concentration, because both blood parameters are indicators of the severity of NEB. However, there was no significant difference between the change in serum NEFA concentrations in both groups. It is thought that changes of BCS during the periparturient could be an indirect parameter for determination of the severity of NEB in dairy cows (Rukkwamsuk, 2010). The present study did not find a correlation between the changes of BCS and the changes of serum NEFA concentrations. However, there was a negative correlation between the changes of BCS during the periparturient period and the decrease of serum glucose concentrations. According to these findings, the changes of BCS during the periparturient period could be used for determination of the severity of NEB in dairy cows. Lactation number The mean lactation number was 1.58 ± 1.84. The distribution of the lactation numbers is depicted in figure 10. The cows were divided in three groups, according to their lactation number: lactation number 0 (n = 9), lactation number 1 (n = 8), lactation number > 1 (n = 9). The means of the blood parameters are depicted in table 2 and compared with the paired samples T-test. No significant differences were found. Wathes et al. (2007) found that primiparous cows had higher NEFA concentrations at calving in comparison with multiparous cows. In the present study, primiparous cows did not have significant higher serum NEFA concentrations immediately after calving. Dairy heifers are generally calved for the first time at about 24 months (Wathes et al. 2007). Heifers are not fully grown at that time, which means that they require energy for their growth. Management and genetic differences between continents can have important effects on the relative maturity at which cows calve for the first time and their response on the first lactation (Wathes et al. 2007). This might explain why the primiparous cows in this study did not have significant higher NEFA concentrations at calving than multiparous cows. The mean change in NEFA concentration did not significantly differ between primiparous and multiparous cows. This indicates that the lactation number has no influence on the severity of NEB immediately after calving. It does not mean that the lactation number has no influence on the severity of NEB in a longer period after calving, because multiparous cows have their peak in serum NEFA concentrations three weeks after parturition.

Figure 10: distribitution of the lactation numbers. 26 cows are included. The mean lactation number was 1.58 ± 1.84. Table 2: means ± SD of the blood parameters
Lactation no. NEFA -4 weeks NEFA 0 weeks ∆NEFA (mmol/L) Glucose -4 weeks Glucose 0 weeks
0 1
(mmol/L)
0.34 ± 0.14 0.40 ± 0.26
(mmol/L)
0.68 ± 0.33 0.78 ± 0.28 0.41 ± 0.39 0.38 ± 0.32
(mg/dL)
54.66 ± 14.30 55.37 ± 8.63
(mg/dL)
40.30 ±10.55 40.15 ± 7.22 >1 0.41 ± 0.20 0.71 ± 0.19 0.30 ± 0.25 53.03 ± 9.12 42.18 ± 8.20 Serum NEFA and glucose concentration A negative correlation between the serum glucose and NEFA concentrations is expected: a decrease in serum glucose concentration will lead to an increase in serum NEFA concentrations. However, there was no significant correlation between the serum glucose en NEFA concentration 4 weeks before calving (r = 0.033) and at week 0 (r = 0.018). There was also no significant correlation between the change in NEFA concentration and the glucose concentration at week 0 (r = 0.005). As mentioned earlier, cows with hypoglycemia did not have a higher change in NEFA concentration. The lack of correlation between the serum NEFA and serum glucose concentration indicates that the cows do not suffer from periparturient health problems associated with a severe NEB, such as fatty liver. Amount of feed The mean amount of concentrate was 5.25 ± 2.86 kg/day and the mean amount of roughage was 23.52 ± 15.76 kg/day. There was no correlation between the amount of concentrate or roughage and the change in NEFA concentration and the serum glucose concentration at week 0. However, the dry matter intake is not recorded in these study and it is not recorded if the cows eat everything. The amount of energy of the diet is also unknown. Analysis of the feedstuff and more accurate data about the actual dry matter intake is necessary. Milk yield The milk yield in the first month of lactation was recorded for 11 cows. The milk yield was recorded one day in each week. The results are provided in table 4. The mean milk yield in week 1 was 15.89 ± 3.01, in week 2 17.64 ± 1.57, in week 3 17.38 ± 2.16 and in week 4 17.3 ± 3.04 (figure 14). There was no significant difference between the mean milk yields. These results are similar with results found in a previous study conducted with Thai dairy cows (Rukkwamsuk 2010).

8 9 11 12 13 14 16 18 The mean change in NEFA concentration was 0.34 ± 0.22 mmol/L between 4 weeks before parturition and week 0. There was no significant correlation between the milk yields and the change in serum NEFA concentration. A negative, significant correlation was expected, because NEB is associated with relatively low milk production. Cows with a higher change in NEFA concentration are expected to have a lower milk yield. This was not found in the present study. The mean glucose concentration was 55.91 ± 8.90 mg/dL 4 weeks before calving and 44.28 ± 6.52 mg/dL at week 0. Table 4: milk yield
Cow ID
1 2 4
Milk yield week 1 (kg)
21.8 10.5 15
Milk yield week 2 (kg)
20.4 19 16
Milk yield week 3 (kg)
19.7 19 15
Milk yield week 4 (kg)
19.5 19.5 15 18 14 14 15.5 18.5 15 18 14.5 18 16 17 15.5 19.5 17 18.4 17.2 18 16 18 14.8 21 15 18.9 15.8 19.5 17 20 15 18.5 10 19.5 16.8 25 20 15 10 5 0 1 2 3 4
Weeks from parturition
Figure 14: the mean milk yield in the first month after parturition. Data represent means and SD as error bars. Cows were divided in two groups, one group with hypoglycemia at week 0 (n = 7) and one group without hypoglycemia at week 0 (n = 4). The glucose concentration at week 0 was significantly lower in the group with hypoglycemia (P = 0.028). The milk yield did not differ significantly between the two groups (figure 16). A significant difference was expected.

Table 5: Group 1 are cows with hypoglycemia.
Group 1 Group 2 Glucose -4 weeks (mg/dL)
54.47 ± 10.55 58.42 ± 5.28
Milk yield week 1 (kg)
16.19 ± 3.61
Glucose 0 weeks (mg/dL)
40.55 ± 4.64 50.79 ± 3.12
Milk yield week 2 (kg)
18.21 ± 1.55
Milk yield week 3 (kg)
17.77 ± 2.46
∆NEFA (mmol/L)
0.31 ± 0.25 0.40 ± 0.15
Milk yield week 4 (kg)
16.97 ± 3.51
Group 1 Group 2
15.38 ± 1.89 16.63 ± 1.11 16.7 ± 1.58 17.88 ± 2.32 Figure 16: Group 1 are cows with hypoglycemia (n = 7), group 2 are cows without hypoglycemia (n = 4). NEFA and milk fever 37 cows were included in this analysis. Their mean serum NEFA concentration 4 weeks before parturition was 0.39 ± 0.17 mmol/L, at week 0 0.68 ± 0.22 mmol/L. The serum NEFA concentration after parturition was significantly higher than the serum NEFA concentration before parturition (P < 0.001). The mean glucose concentration 4 weeks before calving was 53.0 ± 0.06 mg/dL, at week 0 40.92 ± 10.05 mg/dL. The glucose concentration after parturition was significantly lower than before parturition (P < 0.001). This means that the cows suffered from some degree of NEB within 3 days after calving. This is in agreement with the previous analysis conducted in this study with 26 cows. The mean calcium concentration after calving was 8.42 ± 1.72 mg/dL. Two cows in this study had milk fever (serum calcium concentrations < 5.5 mg/dL) and 22 cows had subclinical milk fever (serum calcium concentration between 5.5 and 8.5 mg/dL). There was no significant correlation between the serum glucose concentration and the serum calcium concentration at week 0 (r = 0.1888). There was a significant correlation between the calcium concentration at week 0 and the serum NEFA concentration at week 0 (r = -0.369; correlation is significant at the 0.05 level (2-tailed)). This indicates that cows with a higher serum NEFA concentration at calving had lower serum calcium concentrations at calving. This is in agreement with previous research

(Reinhardt et al. 2010) and indicates that normocalcemic cows have a better energy balance. However, other researchers stated that cows in NEB remain periparturient normocalcemia more effectively than cows in a better energy balance (Roche et al. 2009). The correlation can be explained by the feed intake: cows with a lower feed intake will have higher serum NEFA concentrations and lower serum calcium concentrations. The correlation between the serum NEFA concentration and the serum calcium concentration is depicted in figure 17. Based on the found correlation, it would be expected that more cows suffered from milk fever. However, only 2 cows in the present study suffered from milk fever. This suggests that the calcium intake was high enough to prevent the development of hypocalcemia. Figure 17: scatter plot showing the degree of correlation between the serum NEFA concentration (mmol/L) and the serum calcium concentration (mg/dL). General discussion Only 26 cows were used for this study. This might be an explanation why the results are not in agreement with previous research. Not many cows calf during February and March in Thailand. Not all the cows who had their calving date in March are used for this study, because no data about 4 weeks after calving were available. Cows were randomly selected, which might cause a lack of dispersion and correlations. It is better to select cows in categories, for example cows with a high, normal and low BCS. Many cows included in this study had a low lactation number. Selection of cows according to their lactation number could have caused a better dispersion. Serum NEFA concentrations were measured 0 to 3 days after calving. The concentration of NEFA begins to rise 2 to 4 days before calving and have its peak round 3 days after parturition (Le Blanc 2010). For most cows, it was measured earlier than 3 days after calving, thus before the peak concentration. The change in serum NEFA concentrations might therefore not reflect the true severity of the NEB. Samples should be taken at approximately the same time, due to diurnal or postprandial variations. Just before feeding, the NEFA concentrations peak (LeBlanc 2010). In this study, samples were not taken at the same time. The serum NEFA concentration van be falsely elevated if serum is not separated within 12 to 24 hours of blood collection (Le Blanc 2010). For most samples, serum was separated within this time. However, this is not the case for all the samples.

The BCS was determined by different clinicians. It would be better if one person determined the BCS for all cows. However, farms included in this study did not lie close together and it was not practical to let one clinical determine all BCS. The BCS was for some cows determined one week before calving, for other cows 0 to 3 days after calving. It would be better if the BCS was determined on week 0 for all cows. The BCS is determined in the same way as for Holstein Friesian cows. Thai dairy cows are approximately for 85% Holstein Friesian cows. It might not be correct to determine the BCS with the same scoring system. Feed samples were collected and it was planned to analyze the samples. Due to the limited amount of time in which this study is conducted, the analysis was not finished on time. There were cows with a roughage and concentrate intake of 70 kg a day. This is not possible and might be caused by miscommunation between the veterinarian and the farmer, or wrong translation by the veterinarian.

Conclusion
Thai dairy cows raised in small holder farms suffered from some degree of negative energy balance immediately after calving. This was characterized by loss in body condition score, a decrease of the serum glucose concentration and an increase in serum NEFA concentration. There was no correlation between the NEFA concentration and the change in BCS between 4 weeks before and 4 weeks after parturition. Over-conditioning at calving seemed to be no risk factor for Thai dairy cows, because on average the cows were in optimal condition at calving. There was no correlation between the serum glucose concentration and the serum NEFA concentration. Loss of BCS in the last month of gestation predisposes cows to a more severe NEB immediately after parturition. This is the most relevant risk factor for NEB found in this study.

Acknowledgements
I want to thank dr. J.T. Schonewille for his effort and the opportunity to fulfill my research project outside the Netherlands. I want to thank dr. T. Rukkwamsuk and dr. W. Thiangtum from the Kasetsart University for the opportunity to do my research project at their university and all their help during the project. I want to thank the clinicians from Nong Pho Animal Hospital for their hospitality and their efforts during the project. I have learned a lot from them during my stay in Thailand and the project wouldn’t have succeed without their efforts. I want to thank the other residents of the animal hospital as well. Their kindness and hospitality made my stay at the hospital a wonderful time. I want to thank the farmers of the small-holder farms used in this study.

References
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Sample No.: 1 Farm: Narong Luangkravi Subdistrict: Photaram Province: Rachaburi No. of lactating cows: 16 Cow No.: Chompu Lactation No.: 1 AI date: 3 May 2010 Dry date: 2 December 2010 Calving date: 15 February 2011 Transitional feed date (before calving): When decrease of milk production on dry period, the owner will increase the concentrate feed.
Type of feed (~1 week before calving)
Concentrate: o 16% protein ~ 4 kg/day Roughage: o grass: 6 kg/day o straw: 6 kg/day o silicate (pine-apple): 2 kg/day Remarks: / Mineral supplement before calving: Yes Mineral block: no Vitamin: yes, KT (from radio advertisement) + Catosan, 20 ml Has cow got a milk fever before? No

Sample No.: 5 Farm: Theera Subdistrict: Bangpae Province: Ratchaburi No. of lactating cows: 24 Cow No.: 4 Lactation No.: 5 AI date: 10 May 2010 Dry date: 23 Nov 2010 Calving date: 17 February 2011 Transitional feed date (before calving): 1 month
Type of feed (~1 week before calving)
Concentrate: o NP 20: ~ 0.5*2 kg/day o cassava residue: ~ 5*2 kg/day Roughage: o baby corn: ~ 5*2 kg/day o hay: ~ 4 kg/day Remarks: / Mineral supplement before calving: Yes, Sotext (1 table spoon) Mineral block: no Vitamin: yes, ADE Has cow got a milk fever before? No

Sample No: 7 Farm: Vichai Subdistrict: Tamakha Province: Kanchanaburi No. of lactating cows: 15 Cow No.: Khao phong Lactation No.: 3 AI date: 9 May 2010 Dry date: 22 November 2010 Calving date: 6 February 2011 Transitional feed date (before calving): after dry off for 2 weeks
Type of feed (~1 week before calving)
Concentrate: o NP (16% ep): ~ 3-4 kg/day Roughage: o napier grass: ~ 30 kg/day Remarks: pankota grass Mineral supplement before calving: Yes Mineral block: no Vitamin: yes, Catasal ~ 20 ml + Vit. ADE ~ 15 ml (both 15 days before calving) Has cow got a milk fever before? Yes, 6months ago (2 cows were died)

Sample no.: 9 Farm: Wisoot Sub district: Potaram Province: Ratchaburi No. of lactating cows: 11 Cow No.: Wandee Lactation No.: heifer AI date: 9 May 2010 Dry date: - Calving date: 9 February 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o powder: 3 kg/d o concentrate: 3 kg/d Roughage: o rice straw: 10 kg/d Remarks: cow eat concentrate about 2 kg/day and increase to 4 kg/day when 2 months before calving. Mineral supplement before calving: no Mineral block: yes, SK Best Lick Vitamin: yes, injectavit + catosol Has cow got a milk fever before? No

Sample no.: 10 Farm: Wisoot Sub district: Potaram Province: Ratchaburi No. of lactating cows: 11 Cow No.: Petch Lactation No.: 2 AI date: 1 May 2010 Dry date: 1 December 2010 Calving date: 1 February 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate: 3 kg/d o powder: 3 kg/d Roughage: o rice straw: 10 kg/d Remarks: cow eat concentrate about 2 kg/day when start dry period and increase to 6 kg/day when 1 month before calving. Mineral supplement before calving: no Mineral block: yes, SK Best Lick Vitamin: yes, injectavit + catosol Has cow got a milk fever before? No

Sample no.: 11 Farm: Piw-Ngam Sub district: Potaram Province: Ratchaburi No. of lactating cows: Cow No.: Kaem Lactation No.: heifer AI date: 23 April 2010 Dry date: - Calving date: 30 January 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate: 4 kg/d o pine apple powder: 4 kg/d Roughage: o rice straw: 8 kg/d Remarks: eat concentrate since yearing to heifer about 1 kg/day and increase to 4 kg/day when 3 weeks before calving. Mineral supplement before calving: no Mineral block: no Vitamin: no Has cow got a milk fever before? No

Sample no.: 12 Farm: Piw-Ngam Sub district: Potaram Province: Ratchaburi No. of lactating cows: Cow No.: Phrae Lactation No.: 1 AI date: 22 April 2010 Dry date: 27 October 2010 Calving date: 26 January 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate: 4 kg/d o pine apple powder: 4 kg/d Roughage: o rice straw: 8 kg/d Remarks: eat concentrate about 1 kg/day since start dry period and increase to 3 kg/day when 3 weeks before calving. Mineral supplement before calving: no Mineral block: no Vitamin: yes, ADE Has cow got a milk fever before? No

Sample no.: 13 Farm: Sumran 1 Sub district: Potaram Province: Ratchaburi No. of lactating cows: 18 Cow No.: 70-472957 (1) Lactation No.: 3 AI date: 14 May 2010 Dry date: 5 December 2010 Calving date: 20 February 2011 Transitional feed date (before calving): 5 January 2011
Type of feed (~1 week before calving):
Concentrate: o concentrate pure pride: 2 kg/d o concentrate Nhong Pho: 2 kg/d Roughage: o corn: 10 kg/d o rice straw: 3 kg/d Remarks: cow eat concentrate about 1 kg/day since 1 month before calving. Mineral supplement before calving: yes, about 50 g/day (no brand) Mineral block: no Vitamin: no Has cow got a milk fever before? No

Sample no.: 14 Farm: Sumran 1 Sub district: Potaram Province: Ratchaburi No. of lactating cows: 18 Cow No.: Ree (1) Lactation No.: heifer AI date: 17 May 2010 Dry date: - Calving date: 20 February 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate pure pride: 2 kg/d o concentrate Nhong Pho: 2 kg/d Roughage: o corn: 10 kg/d o rice straw: 3 kg/d Remarks: cow eat concentrate about 1 kg/day since yearing. Mineral supplement before calving: yes, 50 g/day (no brand) Mineral block: no Vitamin: no Has cow got a milk fever before? No

Sample no.: 17 Farm: Outumporn Sub district: Potaram Province: Ratchaburi No. of lactating cows: 27 Cow No.: 08 Lactation No.: 1 AI date: 8 May 2010 Dry date: 6 December 2010 Calving date: 8 February 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate 16% prot: 3 kg/d Roughage: o grass: 10 kg/d o corn: 8 kg/d Remarks: when dry period, cow eat concentrate about 2 kg/day and increase to 4 kg/day 2 weeks before calving. Mineral supplement before calving: no Mineral block: no Vitamin: no Has cow got a milk fever before? No

Sample no.: 18 Farm: Outumporn Sub district: Potaram Province: Ratchaburi No. of lactating cows: 27 Cow No.: 15 Lactation No.: 1 AI date: 25 May 2010 Dry date: 20 December 2010 Calving date: 27 February 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate 16% protein: 3 kg/d Roughage: o grass: 10 kg/d o corn: 8 kg/d Remarks: when dry period, cow eat concentrate about 2 kg/day and increase to 4 kg/day 2 weeks before calving. Mineral supplement before calving: no Mineral block: no Vitamin: no Has cow got a milk fever before? No

Sample no.: 20 Farm: Sumran 2 Sub district: Potaram Province: Ratchaburi No. of lactating cows: 10 Cow No.: Lukesow Lactation No.: AI date: 15 May 2010 Dry date: 11 December 2010 Calving date: 25 February 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate: 8 kg/d Roughage: o straw: 2.5 kg/d o grass: 10 kg/d o silate: 8 kg/d Remarks: when dry period, cow eat concentrate about 2 kg/day and increase to 4 kg/day when 2 weeks before calving. Mineral supplement before calving: no Mineral block: yes, KMZ Vitamin: no Has cow got a milk fever before? No

Sample no.: 21 Farm: Sumran 2 Sub district: Potaram Province: Ratchaburi No. of lactating cows: 10 Cow No.: Lactation No.: heifer AI date: 26 April 2010 Dry date: - Calving date: 30 January 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate: 8 kg/d Roughage: o straw: 2.5 kg/d o grass: 10 kg/d o silate (made of pineapple): 8 kg/d Remarks: cow eat concentrate about 2 kg/day and increase to 4 kg/day 4 weeks before calving. Mineral supplement before calving: no Mineral block: yes, KMZ Vitamin: no Has cow got a milk fever before? No

Sample no.: 22 Farm: Montree Sub district: Potaram Province: Ratchaburi No. of lactating cows: 28 Cow No.: 10 Lactation No.: 1 AI date: 10 May 2010 Dry date: 15 November 2010 Calving date: 14 February 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate: 1 kg/d Roughage: o grass: 20 kg/d o rice straw: 3 kg/d o corn peel: 2 kg/d Remarks: Mineral supplement before calving: yes, KT super selenium plus ½ tablespoon with ½ table spoon salt Mineral block: no Vitamin: no Has cow got a milk fever before? No

Sample no.: 24 Farm: Na-Rong Ku Sub district: Ban-Pong Province: Ratchaburi No. of lactating cows: 20 Cow No.: Som-O Lactation No.: heifer AI date: 21 May 2010 Dry date: - Calving date: 28 February 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate: 2 kg/d Roughage: o corn: 25 kg/d o rice straw: 2 kg/d Remarks: cow eat concentrate since yearing to heifer about 2 kg/day Mineral supplement before calving: no Mineral block; no Vitamin: yes, B12 Has cow got a milk fever before? No

Sample no.: 25 Farm: Wan-Pen Sub district: Ban-Pong Province: Ratchaburi No. of lactating cows: 35 Cow No.: Num-Kang Lactation No.: 1 AI date: 14 May 2010 Dry date: 30 November 2010 Calving date: 20 February 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate: 8 kg/d Roughage: o straw: 8 kg/d o looktao: 5 kg/d Remarks: when dry period, cow eat concentrate about 2 kg/day and increase to 4 kg/day 1 month before calving. Mineral supplement before calving: yes, about 1 table spoon BIO-MIX Mineral block: no Vitamin: yes, phosphonontonic 20% + Fercobsang 12 Has cow got a milk fever before? No

Sample no.: 26 Farm: Wan-Pen Sub district: Ban-Pong Province: Ratchaburi No. of lactating cows: 35 Cow No.: Yod-num Lactation No.: 1 AI date: 14 May 2010 Dry date: 30 November 2010 Calving date: 16 February 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate: 8 kg/d Roughage: o straw: 8 kg/d o looktao: 5 kg/d Remarks: when dry period, cow eat concentrate 2 kg/day and increase to 4 kg/day 1 month before calving. Mineral supplement before calving: yes, about 1 table spoon BIO-MIX Mineral block: no Vitamin: yes, Fercobsang + phosponontonic Has cow got a milk fever before? No

Sample no.: 28 Farm: Wan-Pen Sub district: Ban-Pong Province: Ratchaburi No. of lactating cows: 35 Cow No.: Flower Lactation No.: heifer AI date: 13 May 2010 Dry date: - Calving date: 5 February 2011
Transitional feed date (before calving): Type of feed (~1 week before calving):
Concentrate: o concentrate: 8 kg/d Roughage: o straw: 8 kg/d o looktao: 5 kg/d Remarks: heifer eat concentrate about 2 kg/day since yearing and increase to 4 kg/day 2 months before calving. Mineral supplement before calving: no Mineral block: no Vitamin: yes, Phosphonontonic + Fercobsang Has cow got a milk fever before? No

Sample No.: 29 Farm: Suchada Subdistrict: Tamaka Province: Kanchanaburi No. of lactating cows: 14 Cow No.: Jun Lactation No.: 5 AI date: 01 May 2010 Dry date: 24 November 2010 Calving date: 12 February 2011 Transitional feed date (before calving): 3 weeks after dry off, 2 kg of 20% ep (NP)/day
Type of feed (~1 week before calving)
Concentrate: o 20% ep (NP): ~ 4 kg/day Roughage: o Bawa grass: ~ 32 kg/day o rice straw ~ 5 kg/day Remarks: Mineral supplement before calving: Yes, mineral powder (super selenium) Mineral block: yes Vitamin: yes, catasal 14 days before calving (phosphorour + vit B12 supplement) Has cow got a milk fever before? Yes, 6 years ago

Sample No.: 30 Farm: Khui Subdistrict: Tamakha Province: Kanchanaburi No. of lactating cows: 34 Cow No.: Kularb Lactation No.: 6 AI date: 7 May 2010 Dry date: 3 December 2010 Calving date: 13 February 2011 Transitional feed date (before calving): no concentrate in dry off period until 1 week before calving, then increase to 1 kg
Type of feed (~1 week before calving)
Concentrate: o NP (20% ep): ~ 1 kg/day Roughage: o corn stalk: ~ 10kg/day o (corn stalk ~ 20 kg/day) o silage (corn stalk, pine apple peel, cassava peel) ~ 15 kg/day Remarks: / Mineral supplement before calving: Yes Mineral block: yes Vitamin: yes, catosal Has cow got a milk fever before? Yes, 1 year ago

Sample No.: 31 Farm: Somnuke Subdistrict: Tamakha Province: Kanchanaburi No. of lactating cows: 10 Cow No.: Dok-Oy Lactation No.: 2 AI date: 3 May 2010 Dry date: 29 November 2010 Calving date: 9 February 2011 Transitional feed date (before calving): After dry off feed asith 16% ep (NP) ~ 10,5 kg/day. One month before calving, 16% ep (NP) ~ 4,5 kg. One week before caslving 0,5 kg of 16% ep (NP).
Type of feed (~1 week before calving)
Concentrate: o 16% ep: ~ 0,5 kg/day Roughage: o corn stalk: ~ 25 kg/day or napier grass (if corn cannot harvest) o rice straw: ~ 3 kg/day Remarks: Mineral supplement before calving: Yes Mineral block: No Vitamin: yes, ADE 2 weeks before calving + vit.b12 Has cow got a milk fever before? No
Follow up disease outbreak:
Disease: retent foetal membrane, Date: 10 February 2011

Sample No.: 34 Farm: Somkiat Subdistrict: Tumkang Province: Kanchanaburi No. of lactating cows: 24 Cow No.: Praew Lactation No.: 5 AI date: 8 May 2010 Dry date: 8 November 2010 Calving date: 7 February 2011 Transitional feed date (before calving): 15 days before calving give 20% ep (NP) ~ 2 kg/day + mineral supplement (selenium plus, 0,02%)
Type of feed (~1 week before calving)
Concentrate: o 20% ep (NP): ~ 2 kg/day o Soy bean cake (byproduct from tofu production): ~ 4 kg/day Roughage: o corn stalk: ~ 35-40 kg/day o rice straw: ~ 1 kg/day Remarks: / Mineral supplement before calving: Yes, mineral powder, 15 days before calving Mineral block: no Vitamin: no Has cow got a milk fever before? No

Sample No.: 36 Farm: Sank-petch Subdistrict: Tamakha Province: Kanchanaburi No. of lactating cows: 5 Cow No.: Lay Lactation No.: 2 AI date: 6 May 2010 Dry date: 2 December 2010 Calving date: 14 February 2011 Transitional feed date (before calving): 1 month after dry off, 1 kg/day. Before calving 2,5 kg/day
Type of feed (~1 week before calving)
Concentrate: o 20% ep (NP): ~ 2,5 kg/day Roughage: o corn stalk: ~ 70 kg/day Remarks: / Mineral supplement before calving: Yes Mineral block: yes Vitamin: yes, powder -> goto 1000 (source of vitamin B complex) Has cow got a milk fever before? No

Sample no.: 49 Farm: Sirijun Sub district: Meaung Province: Nakhon Pathom No. of lactating cows: 31 Cow No.: F78 Lactation No.: heifer AI date: 4 June 2010 Dry date: - Calving date: 22 February 2011 Transitional feed date (before calving): yes
Type of feed (~1 week before calving):
Concentrate: o cassava residual: 8 kg/d o concentrate: 2 kg/d Roughage: o rice straw: 6 kg/d o corn chopped: 8 kg/d Remarks: Mineral supplement before calving: no Mineral block: Vitamin: Has cow got a milk fever before? No

Sample no.: 51 Farm: Rungsalit Sub district: Tamakha Province: Kanchanaburi No. of lactating cows: 8 Cow No.: Mini Lactation No.: heifer AI date: 25 May 2010 Dry date: - Calving date: 02 March 2011 Transitional feed date (before calving): 20 days before calving
Type of feed (~1 week before calving):
Concentrate: o 16% ep (NP) ~ 4 kg/day Roughage: o Corn stalk ~ 30 kg/day Remarks: / Mineral supplement before calving: yes, mineral powder (GT1000) Mineral block: yes Vitamin: yes, vitamin ADE Has cow got a milk fever before? No

Sample no.: 54 Farm: Manop Sub district: Tamaka Province: Kanchanaburi No. of lactating cows: 7 Cow No.: Bai Fern Lactation No.: heifer AI date: 31 May 2010 Dry date: - Calving date: 08 March 2011 Transitional feed date (before calving): 1 month before calving (give 1 kg 20% ep NP)
Type of feed (~1 week before calving):
Concentrate: o 20% ep (NP) ~ 4 kg/day Roughage: o Corn stalk ~ 30 kg/day o Or Napier (taniaan) ~ 30 kg/day Remarks: / Mineral supplement before calving: yes, mineral powder (GT1000) Mineral block: yes Vitamin: yes, vitamin ADE Has cow got a milk fever before? No