**3. Use of blood and milk analyses to evaluate nutritional and disease status**

If recognition of subclinical diseases is difficult, the condition may be confirmed by analyzing blood, milk or sometimes urine, although some of them are still difficult to diagnose in practice, including rumen acidosis and fatty liver (Ingvartsen, 2006). Blood and milk analyses as tools to evaluate nutritional and disease status of individual animal or whole herd are aimed to help making decisions for improvement of nutritional strategies and production management. However, many problems are associated with interpretation of results of laboratory analyses, including Compton metabolic profile test, as well as other tools for assessment of metabolic status of dairy animals.

#### **3.1. Milk analyses**

42 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

have retained placenta and metritis (Lewis, 1997).

2006a).

**status** 

endometritis refer to the inflammation of the uterus and of its endometrial lining - both conditions are referred to subsequently as metritis (Urton et al., 2005). Younger cows were more likely to have dystocia or assisted deliveries, while older cows were most likely to

In some herds, up to 40% of the postpartum cows may be diagnosed with, and treated for uterine infections. However, the exact causes of uterine infections are unknown but are associated with several factors. Cows with dystocia, retained placenta, or stillbirths, and other metabolic disorders are more likely to develop metritis than healthy cows. Impaired immune functions before and after calving seem to predispose cows to severe uterine infections. It has been suggested that the function of neutrophils is impaired in cows that develop uterine infections. Thus, methods for regulating immune function in periparturient cows may have potential for preventing uterine infections. However, prevention of uterine infections is still difficult because the primary causes cannot be defined clearly (Lewis, 1997). Malnutrition influences the ability of the immune system to function, which affects the incidence of diseases such as mastitis and metritis (Goff,

Cows suffering metritis exhibit reduced milk yield and reproductive performances (Urton et al., 2005). Few cows die from uterine infections, but affected animals are more likely to be culled for poor reproductive performances. The estimated cost to producers for each cow with metritis was 106 U.S. dollars (Lewis, 1997). Many of the financial costs of metritis are indirect, such as increased days open or predisposition to other diseases, and are thus difficult to measure. As mentioned previously, metritis is one of the diseases that predispose

In contrast, the effects of metritis on milk production can be measured immediately, and losses during first four months after calving can be almost 270 kg. Also, besides reduced milk production in sick animals, some pharmaceuticals for treatment of uterine infections contaminate milk with residues, and the milk must be discarded. This is how uterine

**3. Use of blood and milk analyses to evaluate nutritional and disease** 

If recognition of subclinical diseases is difficult, the condition may be confirmed by analyzing blood, milk or sometimes urine, although some of them are still difficult to diagnose in practice, including rumen acidosis and fatty liver (Ingvartsen, 2006). Blood and milk analyses as tools to evaluate nutritional and disease status of individual animal or whole herd are aimed to help making decisions for improvement of nutritional strategies and production management. However, many problems are associated with interpretation of results of laboratory analyses, including Compton metabolic profile test, as well as other

dairy cows to foot problems and left displaced abomasums (Shaver, 1997).

infections may have an indirect effect on milk production (Lewis, 1997).

tools for assessment of metabolic status of dairy animals.

Producers can use changes in milk production or in milk composition to monitor the health of their animals, but these tools are not always completely reliable because milk production corresponds poorly with mild or subclinical infectious disease. Nevertheless, measuring energy balance from changes in milk composition, most likely changes in milk fat and protein contents, could provide a cheap and reliable estimator of energy balance. A study clearly showed that there was a strong relationship between energy balance and milk composition under stable feeding conditions (Friggens et al., 2007). Milk composition varies with energy status and was proposed for measuring energy balance on-farm. Ratio milk fat/protein below 1,4 indicates the optimal or positive energy balance, and above it balance is negative (Pehrson, 1996; Zadoks, 2006). During peak lactation many cows with negative energy balance have this ratio in milk even above 2,1 (Pehrson, 1996).

The biological basis for the relationship between fat/protein ratio and energy status of the animal can be found in two physiological features: a) mechanism that maintains milk energy output by increasing milk fat content when yield is compromised due to a deficit in energy supply, and b) decreased milk protein content under negative energy balance (Friggens et al., 2007). Negative energy balance also may be reflected in elevated ketone bodies which are excreted in milk and urine. Milk has about half the ketone level of blood, and is recommended to check milk ketone levels for detection of ketosis.

Milk fat depression is an example of disorder that can be diagnosed exclusively by milk analysis. Specific blood test for MFD diagnosis does not exists, but milk analyses are usually enough to evaluate risk for or presence of MFD in a herd. Every drop in milk fat percentages with no changes in the content of other milk constituents and milk yield can be considered as MFD. In serious cases, diet-induced MFD can result in reduction of milk fat yield of up to 50% or even more (Bauman & Griinari, 2001; Bauman et al., 2008) and represents one of the greatest risks for production economy.

### **3.2. Blood analyses**

Analyses of blood constituents have long been a matter of concern as a possible tool for assessment of diet adequacy or metabolic and health status of animals. Compton metabolic profile test (CMPT) originally involved the analysis of a set of blood variables from three groups of seven cows, one near peak lactation, and others in midlactation and in late dry period (Kronfeld et al., 1982). The samples are collected at least three times yearly: summer, autumn and winter, or when nutritional imbalance is expected, using the same procedures and timings (Radostits et al., 2000). Means of variables are calculated for each lactational group in each herd, and this set of means constitutes the metabolic profile. Each group mean is compared to reference ranges determined from corresponding means plus or minus two standard deviations that are based on group-means for all herds (Kronfeld et al., 1982). The test was based on the concept that laboratory measurement of certain components of the blood will reflect the nutritional status of an animal with or without presence of clinical abnormalities. However, the results of research indicate that CMPT may be useful only as an

aid in the diagnosis of the nutritional imbalances and production diseases. It must be carefully planed and is still expensive. Laboratories with automated analytical equipment should be available and this is often a major limiting factor (Radostits et al., 2000).

Compton metabolic profile test includes following analyses: blood glucose, packed cell volume, hemoglobin, blood urea nitrogen, serum inorganic phosphate, serum Ca, Mg, K and Na, total serum protein, albumin and globulins, serum Cu and iron (Fe), and plasma NEFA. Obtained results are interpreted with other relevant information taken on the day of sampling related to individual animals or to the herd: age, milk yield, days in milk, concentrates and forage intake, etc (Radostits et al., 2000).

Workers tested the CMPT as a guide to the nutritional status of dairy cows, and found that blood variables were not reliable predictors of energy and nutrients consumption relative to the requirements. Then they supposed that, for prediction of nutritional status, selection of blood variables should be probably different. Those workers have suggested, however, that protein intake may be reflected in blood concentrations of urea, albumin, and hemoglobin. Similarly, blood glucose concentration has attracted attention as an index of energy intake, although results from study to study have been inconsistent (Kronfeld et al., 1982).

Despite limitations and unconformity of metabolic profile tests, blood analyses still can help in diagnostics of some nutritional imbalances. Negative energy balance in postpartum cows can be detected by change in concentration of some blood metabolites (Rushen et al., 2008). As condition that affect immune functions and predispose animals to many metabolic disorders, it is important that negative energy balance is reflected in a higher levels of NEFA and beta-hydroxybutyric acid (BHBA) in the blood. To screen a herd for negative energy balance, Zadoks (2006) recommends having at least 12 animals tested. Testing of NEFA is done 2-14 days before calving and BHBA testing at 2-21 days after calving. If more than 10- 15% of animals have NEFA levels above 0,40 mEq/L or BHBA levels above 1,36 mmol/L, the herd is considered to be suffering from excessive negative energy balance. Ketosis and fatty liver are diseases typically associated with negative energy balance for long periods of time. Increased ketone bodies and NEFA in the blood, and decreased blood glucose level are common findings in cows with ketosis and fatty liver. Values of glucose below 2,2 mmol/L are considered subnormal. However, fatty liver in cows currently can be diagnosed only by liver biopsy (Bobe et al., 2004).

The concentration of BHBA in the blood may be taken as a guide for correction of ration supplement for ewes during the final weeks of pregnancy to prevent pregnancy toxemia. It has been recommended to check 10% of the flock and feed is increased if blood concentration of BHBA exceeds 0,8 mmol/L (Morgante, 2004). Besides hypoglycemia and hyperketonaemia, plasma cortisol levels also may be elevated in sheep with pregnancy toxemia (Andrews et al., 1996). Early in the disease, both does and ewes will show a positive test for ketone bodies in the urine (Morgante, 2004).

There are no specific blood variables for detecting metabolic diseases like laminitis, displaced abomasums, udder edema, retained placenta and metritis. However, if these diseases are herd problems, blood metabolic profile should be checked for more than one variable, and carefully used as an aid for development of preventive strategies. When laminitis is a problem, a metabolic profile on both dry and lactating cows as well as springing heifers should include both red and white blood cells count, packed cell volume, Se, Zn, Cu, Fe, blood urea nitrogen, vitamins A and E, and beta-carotene. When retained placenta is a herd problem, blood profile should include serum minerals, Se, vitamin E, and beta-carotene. In individual cases, blood urea nitrogen and packed cell volume should be included. Metritis requires testing metabolic profile on dry and fresh cows which should include white blood cells count, Se, Zn, Cu, Fe, Mg, blood urea nitrogen, vitamins A and E, and beta-carotene.

44 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

concentrates and forage intake, etc (Radostits et al., 2000).

liver biopsy (Bobe et al., 2004).

test for ketone bodies in the urine (Morgante, 2004).

aid in the diagnosis of the nutritional imbalances and production diseases. It must be carefully planed and is still expensive. Laboratories with automated analytical equipment

Compton metabolic profile test includes following analyses: blood glucose, packed cell volume, hemoglobin, blood urea nitrogen, serum inorganic phosphate, serum Ca, Mg, K and Na, total serum protein, albumin and globulins, serum Cu and iron (Fe), and plasma NEFA. Obtained results are interpreted with other relevant information taken on the day of sampling related to individual animals or to the herd: age, milk yield, days in milk,

Workers tested the CMPT as a guide to the nutritional status of dairy cows, and found that blood variables were not reliable predictors of energy and nutrients consumption relative to the requirements. Then they supposed that, for prediction of nutritional status, selection of blood variables should be probably different. Those workers have suggested, however, that protein intake may be reflected in blood concentrations of urea, albumin, and hemoglobin. Similarly, blood glucose concentration has attracted attention as an index of energy intake,

Despite limitations and unconformity of metabolic profile tests, blood analyses still can help in diagnostics of some nutritional imbalances. Negative energy balance in postpartum cows can be detected by change in concentration of some blood metabolites (Rushen et al., 2008). As condition that affect immune functions and predispose animals to many metabolic disorders, it is important that negative energy balance is reflected in a higher levels of NEFA and beta-hydroxybutyric acid (BHBA) in the blood. To screen a herd for negative energy balance, Zadoks (2006) recommends having at least 12 animals tested. Testing of NEFA is done 2-14 days before calving and BHBA testing at 2-21 days after calving. If more than 10- 15% of animals have NEFA levels above 0,40 mEq/L or BHBA levels above 1,36 mmol/L, the herd is considered to be suffering from excessive negative energy balance. Ketosis and fatty liver are diseases typically associated with negative energy balance for long periods of time. Increased ketone bodies and NEFA in the blood, and decreased blood glucose level are common findings in cows with ketosis and fatty liver. Values of glucose below 2,2 mmol/L are considered subnormal. However, fatty liver in cows currently can be diagnosed only by

The concentration of BHBA in the blood may be taken as a guide for correction of ration supplement for ewes during the final weeks of pregnancy to prevent pregnancy toxemia. It has been recommended to check 10% of the flock and feed is increased if blood concentration of BHBA exceeds 0,8 mmol/L (Morgante, 2004). Besides hypoglycemia and hyperketonaemia, plasma cortisol levels also may be elevated in sheep with pregnancy toxemia (Andrews et al., 1996). Early in the disease, both does and ewes will show a positive

There are no specific blood variables for detecting metabolic diseases like laminitis, displaced abomasums, udder edema, retained placenta and metritis. However, if these diseases are herd problems, blood metabolic profile should be checked for more than one

although results from study to study have been inconsistent (Kronfeld et al., 1982).

should be available and this is often a major limiting factor (Radostits et al., 2000).

Use of blood analyses to evaluate degree of hypocalcaemia is sufficient diagnostic test for this disorder. Concentration of Ca may be determined in whole blood, but more often determination is performed in plasma or serum. When there are no disturbances in acidbase status and protein metabolism, ionized and total Ca are strongly correlated and therefore total Ca concentrations in plasma may serve as an acceptable diagnostic value (Oetzel, 1988).

Hypocalcaemia means that the content of total Ca in the blood is below 2 mmol/L, or below 1 mmol/L of ionized Ca (Massey et al., 1993; Oetzel, 1996). Signs of paresis can occur at total Ca level of 1,9 mmol/L, but most of the cows remain on their feet down to 1,0-1,25 mmol/L. A reduction in the plasma Ca concentration at parturition is usually accompanied by hypophosphataemia and hypermagnesaemia (Phillippo et al., 1994). However, this is not always the case, especially if hypocalcaemia is not severe and is not clinically manifested (Crnkic et al., 2010; Joyce et al., 1997; Oetzel et al., 1988).

Blood analysis is also sufficient diagnostic test for hypomagnesaemia. Based on blood serum or plasma Mg levels, animals may be normally magnesemic (0,74-1,23 mmol/L), chronically hypomagnesemic (0,20-0,74 mmol/L) or acutely hypomagnesemic (<0,20 mmol/L) (Mayland, 1988). Sampling and analysis of the blood of several cows within 12 hours after calving is a good indicator of Mg status of the periparturient period. If serum Mg concentrations are less than 0,82 mmol/L in 90% of cows it suggests insufficient dietary Mg absorption (Goff, 2006a).

## **4. Nutritional strategies to reduce metabolic diseases incidences**

Although metabolic disorders are not easily categorized as to their cause, nutritional strategies have been developed to help prevent many of these disorders (Goff, 2006a). Scientific justification of those measures can be found in many research articles published in scientific journals during the last decades. However, problems in application of the strategies in the field still exist, as well as their limitations and possible negative consequences on milk production economy in certain circumstances. Strategies are based on major factors directly or indirectly increasing the risk of diseases such as overconditioning at calving, excessive mobilization of body fat, low nutrient intake, nutrient or diet specific factors and management and environmental stress (Ingvartsen, 2006). Strategies are expected to be able to reduce morbidity and at the same time improve reproduction and production.

To prevent metabolic disorders in the periparturient period nutritional strategies must start prior to calving. In a survey conducted by Curtis et al. (1985) consumption of nutrients before calving was directly related to the occurrence of metabolic disorders, and directly or indirectly to the occurrence of reproductive disorders after parturition. In most cases, these disorders occur as a complex and many of them are interrelated in their occurrence (Beede, 1995; Curtis et al., 1985). Consequently, strategies to reduce one disease can help preventing others. For example, strategies to reduce liver triglyceride accumulation at calving may decrease incidence of ketosis, etc.

Goff & Horst (1997c) recommended that three basic physiological functions must be maintained during the periparturient period if disease is to be avoided: adaptation of the rumen to lactation diets that are high in energy density, maintenance of a strong immune system, and maintenance of normocalcaemia. Whenever one or more of these functions are impaired, the incidence of both metabolic and infectious diseases is increased. However, measures to maintain these physiological functions must be conducted carefully avoiding other risk factors such as overconditionig and excessive mobilization of body fat around calving.

Metabolic disorders that may appear in the rest of lactation also require adequate prevention strategy to be controlled, including rumen acidosis, laminitis, hypomagnesaemia and milk fat depression.

## **4.1. Adaptation of the rumen**

Fully adapting the rumen flora to a high starch diet that will be fed after parturition requires about 3 to 4 weeks, and full development of rumen papillae requires about 5 weeks of concentrate feeding (Goff & Horst, 1997c). It is, therefore, important to start increasing concentrates in the diet 3-4 weeks before calving and continue during the first 1-2 weeks after parturition to fully adapt rumen to lactation diet. If fresh cow is abruptly switched to a high starch lactation diet, the risk of developing rumen acidosis exists because the lactate production and accumulation. During the rest of lactation, the most reliable means of preventing rumen acidosis is to apply feeding methods that ensure a more even distribution of feed intake over the day. Feeding TMR only once or twice a day may result in that cows eat an excessive amount of feed during a short period of time, because cows are strongly attracted by the arrival of fresh food (Rushen et al., 2008). Buffering agents such as Nabicarbonate or alkalinizing agents such as Mg-oxide are added to high concentrate ration to reduce the risk of acidosis (Goff, 2006a). Na-bicarbonate should be supplemented particularly to corn silage-based diets at the rate of approximately 0,8 to 1% of DM (Stone, 2004). Too rapid increase in concentrate allowance during early lactation may reduce roughage intake and increase the risk of not only rumen acidosis, but also displaced abomasums (Ingvartsen, 2006).

A highly significant relationship between forage neutral detergent fiber (NDF) content in the diet and ruminal pH has been found. NRC (2001) recommended 19% of forage NDF as absolute minimum when formulating rations in the field. A system of "physically effective" NDF (peNDF) relates the ability of a feedstuff to stimulate chewing relative to a hypothetical long grass hay containing 100% NDF. The peNDF of a feed is the product of its physically effective fiber (pef) and NDF content. The diet should contain about 22% peNDF to maintain ruminal pH of 6,0 (Stone, 2004).

46 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

decrease incidence of ketosis, etc.

calving.

and milk fat depression.

**4.1. Adaptation of the rumen** 

abomasums (Ingvartsen, 2006).

To prevent metabolic disorders in the periparturient period nutritional strategies must start prior to calving. In a survey conducted by Curtis et al. (1985) consumption of nutrients before calving was directly related to the occurrence of metabolic disorders, and directly or indirectly to the occurrence of reproductive disorders after parturition. In most cases, these disorders occur as a complex and many of them are interrelated in their occurrence (Beede, 1995; Curtis et al., 1985). Consequently, strategies to reduce one disease can help preventing others. For example, strategies to reduce liver triglyceride accumulation at calving may

Goff & Horst (1997c) recommended that three basic physiological functions must be maintained during the periparturient period if disease is to be avoided: adaptation of the rumen to lactation diets that are high in energy density, maintenance of a strong immune system, and maintenance of normocalcaemia. Whenever one or more of these functions are impaired, the incidence of both metabolic and infectious diseases is increased. However, measures to maintain these physiological functions must be conducted carefully avoiding other risk factors such as overconditionig and excessive mobilization of body fat around

Metabolic disorders that may appear in the rest of lactation also require adequate prevention strategy to be controlled, including rumen acidosis, laminitis, hypomagnesaemia

Fully adapting the rumen flora to a high starch diet that will be fed after parturition requires about 3 to 4 weeks, and full development of rumen papillae requires about 5 weeks of concentrate feeding (Goff & Horst, 1997c). It is, therefore, important to start increasing concentrates in the diet 3-4 weeks before calving and continue during the first 1-2 weeks after parturition to fully adapt rumen to lactation diet. If fresh cow is abruptly switched to a high starch lactation diet, the risk of developing rumen acidosis exists because the lactate production and accumulation. During the rest of lactation, the most reliable means of preventing rumen acidosis is to apply feeding methods that ensure a more even distribution of feed intake over the day. Feeding TMR only once or twice a day may result in that cows eat an excessive amount of feed during a short period of time, because cows are strongly attracted by the arrival of fresh food (Rushen et al., 2008). Buffering agents such as Nabicarbonate or alkalinizing agents such as Mg-oxide are added to high concentrate ration to reduce the risk of acidosis (Goff, 2006a). Na-bicarbonate should be supplemented particularly to corn silage-based diets at the rate of approximately 0,8 to 1% of DM (Stone, 2004). Too rapid increase in concentrate allowance during early lactation may reduce roughage intake and increase the risk of not only rumen acidosis, but also displaced

A highly significant relationship between forage neutral detergent fiber (NDF) content in the diet and ruminal pH has been found. NRC (2001) recommended 19% of forage NDF as absolute minimum when formulating rations in the field. A system of "physically effective" Gradual adjustment of the rumen to a high concentrate diet can help avoiding serious drop in feed intake around calving and minimize body fat mobilization in early lactation. The plasma NEFA concentration is negatively correlated with DMI and depression in feed intake around the time of calving was largely responsible for fatty liver development (Goff, 2006a). Therefore, all nutritional measures that prevent drop in DMI before parturition may be useful in prevention of fatty liver. However, it has been assumed that increased energy and nutrient density of the diet may assure maintenance of the same intake of nutrients and energy despite lower DMI around calving, and decrease rate of lipid mobilization. Increase in nutrient density during the last 2–3 weeks prepartum by increasing concentrates in the ration has been referred to as "steaming-up" or "close-up" diet (Ingvartsen, 2006). Use these diets should not last for too long because of risk of overfeeding energy and development of obesity. NRC (2001) recommends increasing the energy content of the precalving diet from 5,2 MJ/kg of DM during the "far-off" dry period, to 6,8 MJ/kg of DM for the 3 weeks before calving. This strategy is thought to prepare the cow for the metabolic demands of early lactation and thereby minimize the need for body tissue mobilization (Urton et al., 2005).

As fatty liver and ketosis in cows often occur as a complex, all measures to reduce fatty liver incidence may decrease incidence of ketosis (Grummer, 1993). Moreover, positive effects on ketosis incidence and lipid transport also have been seen when niacin or rumen-protected choline is fed to dry and fresh cows (Goff, 2006a). The treatment of pregnancy toxemia in ewes is usually unsuccessful, therefore the prevention is of key importance to reduce occurrence of the disease. Grain is a high source of available energy. Feeding 0,5-1 kg of grain daily along with high quality hay during the last four to six weeks of pregnancy will help prevent pregnancy toxemia.

Manipulation of the nutritional program of dairy cows affects rumen health, which influences hoof health (Stone, 2004). It is very evident that feeding diets that cause drop in rumen pH will result in increase in laminitis cases. To minimize the drop in rumen pH it is necessary to limit amount of concentrate fed per meal to no more than 3,6 kg and provide fresh feed at the bunk throughout the day. Diet should contain at least 25% total NDF or 19% acid detergent fiber (ADF), with non-fiber carbohydrate (NFC) levels between 35 and 40% or non-structural carbohydrate (NSC) levels between 30 to 35%, and minimize abrupt changes of ration. Adding buffers to diets may help maintain claw integrity as buffers, such as Na-bicarbonate, minimize the drop in rumen pH. Supplements that contain combinations of complexed trace minerals Zn, Mn, Cu and Co positivelly influence claw health, and it has been advised to feed them when lameness is problem in a herd.

Increased energy content of the diet fed during the prepartum period was also associated with decreased incidence of displaced abomasum (Curtis et al., 1985). Risk of displaced abomasum for cows fed silage can be almost eliminated if every cow eats a kilogram of straw daily (Ingvartsen, 2006).

## **4.2. Maintenance of immunity**

Maintaining of a strong immune system may help to reduce incidence of infectious diseases after parturition including those related to nutritional factors: retained placenta, metritis, mastitis, laminitis, etc. Inadequate nutrition may contribute depression of the immune system that occurs around calving time (Rushen et al., 2008) when stress of parturition and metabolic challenges experienced by the dairy cow at the onset of milk production impair immune cell function (Goff, 2006a).

Dietary factor that may influence immune functions include vitamin E and Se as the most important vitamin and mineral related to immunity. To a lesser extent vitamin A and betacarotene, Cu and Zn may play a role (Zadoks, 2006). Metabolic conditions related to parturition also may exacerbate immunosuppression, including negative energy balance (Zadoks, 2006), hypocalcaemia (Ganjkhanlou et al., 2010), and hyperketonemia (Overton & Waldron, 2004). Therefore, strategies to maintain strong immune system must include feeding adequate or increased levels of all dietary components that influence immunity, as well as prevention of metabolic disorders that may exacerbate immunosuppression.

Most measures explained in the preceding section related to adaptation of the rumen to lactation diets may help maintain immune functions throughout reduction in negative energy balance and hyperketonemia. Measures to reduce hypocalcaemia around calving are presented in the following section. Feeding vitamin E and Se have long been associated with reducing incidences of retained placentas and uterine infections. Increased protein content of the diet fed during the prepartum period was also associated with decreased incidences of retained placenta (Curtis et al., 1985).

Although feeding is generally not related to the incidence and severity of udder edema, some dietary measures can help prevention of the problem. Restriction of the salt NaCl in the diet reduces the severity of udder edema (Randalu et al., 1974). Restriction or exclusion of other Na or K sources from the diet is also recommended until the levels of dietary Na and K are not in line with dietary recommendations. NRC (2001) recommends at least 0,1% Na in dietary DM for close-up dry cow diet and at least 1% K. As membrane permeability may be a primary cause of udder edema, vitamin E supplementation precalving may be a supportive preventive measure (Thomas et al., 1990). More recently, feeding anionic diets or diets with additional antioxidants have shown some promise in reducing udder edema (Goff, 2006a). Prevention of udder edema is important as condition has temporary effects of pain and stress for the cow, and may increase risk of udder inflammation and additional health problems.

## **4.3. Prevention of parturient hypocalcaemia**

Concerning effects of hypocalcaemia on many physiological functions it is obvious that health, production and reproduction can be compromised in hypocalcemic cows even in the absence of clinical signs of paresis (Oetzel, 1996). Therefore, measures to prevent decline of blood Ca in cows after calving can improve milk production in herds that apparently have no problem with this disease. Prevention aims to increase the mobilization of Ca from the skeleton, or its absorption in the gastrointestinal tract, or both. The most important nutritional measures for prevention of hypocalcaemia include manipulation of minerals in the diet which is nutritionally balanced in accordance with requirements of the cows several weeks before calving. This method of prevention is based on limiting the consumption of Ca or increasing the content of anions in the diet.

#### *4.3.1. Anionic diet*

48 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

Maintaining of a strong immune system may help to reduce incidence of infectious diseases after parturition including those related to nutritional factors: retained placenta, metritis, mastitis, laminitis, etc. Inadequate nutrition may contribute depression of the immune system that occurs around calving time (Rushen et al., 2008) when stress of parturition and metabolic challenges experienced by the dairy cow at the onset of milk production impair

Dietary factor that may influence immune functions include vitamin E and Se as the most important vitamin and mineral related to immunity. To a lesser extent vitamin A and betacarotene, Cu and Zn may play a role (Zadoks, 2006). Metabolic conditions related to parturition also may exacerbate immunosuppression, including negative energy balance (Zadoks, 2006), hypocalcaemia (Ganjkhanlou et al., 2010), and hyperketonemia (Overton & Waldron, 2004). Therefore, strategies to maintain strong immune system must include feeding adequate or increased levels of all dietary components that influence immunity, as

Most measures explained in the preceding section related to adaptation of the rumen to lactation diets may help maintain immune functions throughout reduction in negative energy balance and hyperketonemia. Measures to reduce hypocalcaemia around calving are presented in the following section. Feeding vitamin E and Se have long been associated with reducing incidences of retained placentas and uterine infections. Increased protein content of the diet fed during the prepartum period was also associated with decreased incidences

Although feeding is generally not related to the incidence and severity of udder edema, some dietary measures can help prevention of the problem. Restriction of the salt NaCl in the diet reduces the severity of udder edema (Randalu et al., 1974). Restriction or exclusion of other Na or K sources from the diet is also recommended until the levels of dietary Na and K are not in line with dietary recommendations. NRC (2001) recommends at least 0,1% Na in dietary DM for close-up dry cow diet and at least 1% K. As membrane permeability may be a primary cause of udder edema, vitamin E supplementation precalving may be a supportive preventive measure (Thomas et al., 1990). More recently, feeding anionic diets or diets with additional antioxidants have shown some promise in reducing udder edema (Goff, 2006a). Prevention of udder edema is important as condition has temporary effects of pain and stress for the cow, and may increase risk of udder inflammation and additional

Concerning effects of hypocalcaemia on many physiological functions it is obvious that health, production and reproduction can be compromised in hypocalcemic cows even in the absence of clinical signs of paresis (Oetzel, 1996). Therefore, measures to prevent decline of blood Ca in cows after calving can improve milk production in herds that apparently have

well as prevention of metabolic disorders that may exacerbate immunosuppression.

**4.2. Maintenance of immunity** 

immune cell function (Goff, 2006a).

of retained placenta (Curtis et al., 1985).

**4.3. Prevention of parturient hypocalcaemia** 

health problems.

An anionic, or acidic, diet is one supplemented with anionic salts to provide more anions (Cl and S2-) relative to the cations (Na+ and K+). It has a negative dietary cation-anion difference (DCAD), calculated as mEq/kg of DM = (Na + K) – (Cl + S). It is well known that anionic diets help maintain blood Ca at parturition and prevent milk fever when fed to cows during the last several weeks of pregnancy (Block, 1984). Feeding an anionic diet before parturition has been advised if the incidence of milk fever in a herd exceeds 10%, and also when it is desired to improve the health status and production in herds in which MF is not a serious problem (Horst et al., 1994; Oetzel, 1993).

Diet with excess of anions, mainly Cl- and S2-, relative to the cations Na+ and K+ acidifies the body and is therefore considered as acidogenic (Goff & Horst, 1997b). The two PTHdependent functions, including bone resorption and production of 1,25-(OH)2D, were enhanced after feeding acidogenic diet which contributes to lower incidence of hypocalcaemia after calving (Goff, 2000). However, the diets of dry cows in the field are always more or less alkalogenic and have a positive DCAD. When feeding diets with positive DCAD the acid-base balance tends to metabolic alkalosis, and vice versa. Formulation of acidogenic diet is only possible by supplementation with chloride and sulfate in a quantity that provides a relative excess of Cl- and S2- in the diet. Manipulation of dietary cations and anions, however, is limited by metabolic requirements and by tolerance levels of minerals in the diet.

The literature offered a dozen different formulas to calculate DCAD, which include some or all dietary minerals with or without the use of coefficients related to their utilization from the diet or the degree of influence on acid-base status:


Equations A, B, and C imply the complete absorption of each dietary element that can be considered accurate only in the case of equation C. Equations E and G take into account the coefficients of utilization of those elements that are not completely utilized. Equations H and I could be considered biologically and functionally most accurate because they include the degree of influence of individual ions on acid-base status, and are physiologically the most relevant (Horst & Goff, 1997; Goff, 2000).

Equation F can be used to assess the risk of developing MF. All the elements that increase the risk of MF are on the left side, while those who decrease the risk are on the right side of equation. Equation D can serve the same purpose (Goff et al., 1991). The last equation J, (Na + K) − (Cl + 0,6S), was the most recently proposed by Goff et al. (2004).

Oetzel (1991) collected data from 75 published experiments and determined the risk factors of diet in the development of MF using meta-analysis technique. Comparing the three different equations to calculate DCAD he found that the equation (Na + K) - (Cl + S) was strongly correlated with the appearance of MF. The author in the same study well justified the inclusion of S in the calculation of DCAD. Charbonneau et al. (2006) used the same technique but found that the equation (Na + K) − (Cl + 0,6S) was the most highly associated with clinical milk fever (R2 = 0,44) and urinary pH (R2 = 0,85).


**Table 1.** Mineral elements in the diet used for calculation of DCAD

Mineral content in feed is usually expressed as a percentage so it is necessary to convert it in mEq/kg for calculation of DCAD. Equivalent weight (g/Eq) of each element is calculated by dividing the relative atomic weight (Ar) with its valence. For example, Ar of S is 32,06. Dividing 32,06 by 2 we get the equivalent weight of S which is 16,03 g/Eq. Factor for conversion of percentage in mEq/kg is calculated by dividing the 10.000 with equivalent weight of the element. For S it is 10.000/16,03 = 623,83 (Table 1). Multiplying this factor with percentage of the element in dietary DM its content is expressed in mEq/kg DM. When this is calculated for each element in the equation, then the sum of cations is subtracted from the sum of anions and DCAD value is obtained. The example of calculating DCAD of alfalfa hay and rapeseed meal using equation (Na + K) - (Cl + S) is given in Table 2.


**Table 2.** Example of calculating DCAD in alfalfa hay and rapeseed meal

50 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

+ K) − (Cl + 0,6S), was the most recently proposed by Goff et al. (2004).

with clinical milk fever (R2 = 0,44) and urinary pH (R2 = 0,85).

Element Ar Valence Equivalent weight

**Table 1.** Mineral elements in the diet used for calculation of DCAD

and rapeseed meal using equation (Na + K) - (Cl + S) is given in Table 2.

relevant (Horst & Goff, 1997; Goff, 2000).

Equations A, B, and C imply the complete absorption of each dietary element that can be considered accurate only in the case of equation C. Equations E and G take into account the coefficients of utilization of those elements that are not completely utilized. Equations H and I could be considered biologically and functionally most accurate because they include the degree of influence of individual ions on acid-base status, and are physiologically the most

Equation F can be used to assess the risk of developing MF. All the elements that increase the risk of MF are on the left side, while those who decrease the risk are on the right side of equation. Equation D can serve the same purpose (Goff et al., 1991). The last equation J, (Na

Oetzel (1991) collected data from 75 published experiments and determined the risk factors of diet in the development of MF using meta-analysis technique. Comparing the three different equations to calculate DCAD he found that the equation (Na + K) - (Cl + S) was strongly correlated with the appearance of MF. The author in the same study well justified the inclusion of S in the calculation of DCAD. Charbonneau et al. (2006) used the same technique but found that the equation (Na + K) − (Cl + 0,6S) was the most highly associated

(g/Eq)

Mineral content in feed is usually expressed as a percentage so it is necessary to convert it in mEq/kg for calculation of DCAD. Equivalent weight (g/Eq) of each element is calculated by dividing the relative atomic weight (Ar) with its valence. For example, Ar of S is 32,06. Dividing 32,06 by 2 we get the equivalent weight of S which is 16,03 g/Eq. Factor for conversion of percentage in mEq/kg is calculated by dividing the 10.000 with equivalent weight of the element. For S it is 10.000/16,03 = 623,83 (Table 1). Multiplying this factor with percentage of the element in dietary DM its content is expressed in mEq/kg DM. When this is calculated for each element in the equation, then the sum of cations is subtracted from the sum of anions and DCAD value is obtained. The example of calculating DCAD of alfalfa hay

Na 22,99 1 22,99 434,98 K 39,09 1 39,09 255,77 Ca 40,08 2 20,04 499,00 Mg 24,31 2 12,16 822,88 Cl 35,45 1 35,45 282,06 S 32,06 2 16,03 623,83 P 30,97 3 10,32 968,56

Conversion factor (from % in mEq/kg) The example in Table 2 clearly shows the difference in the alkali-acid production potential of the two feeds depending on their mineral content. A positive DCAD of alfalfa hay is result of the relatively high K content. Extremely negative DCAD of rapeseed meal is result of high S content.

Most diets commonly used for dry cows have DCAD value of +100 to +250 mEq/kg DM (Oetzel, 2000) and even up to +500 mEq/kg DM (Pehrson et al., 1999). By choosing appropriate feeds DCAD can be reduced, but not sufficiently to influence Ca metabolism. It is therefore necessary to enrich the diet with Cl and S by adding anionic salts in a quantity that provides a negative DCAD. That is, indeed, a standard diet that contains all essential nutrients required by dry cow and was further enriched with sources of Cl and S in amount that causes a mild metabolic acidosis.

Acidogenic, or anionic mineral salts are chlorides and sulfates with a high content of Cl and S, not containing Na and K. Alkalogenic salts are, on the other hand, Na and K salts that contain organic anion which is metabolized and are also called cationic salts (Na2CO3, NaHCO3, K2CO3, KHCO3, etc). They have the effect opposite to anionic salts and are undesirable in the diet of dry cows.

To formulate an anionic diet three chlorides and three sulfates are commonly used (Table 3). Two or more of them are usually added depending on mineral content of the diet. Ca and Mg salts also serve to meet requirements in these two minerals whose content in anionic diet should be higher than in standard diet (NRC, 2001). Ammonium salts, besides Cl and S, are also a source of non-protein nitrogen.

Acid production potential or acidifying activity of certain salt depends on preferential absorption of anions in relation to cations which the salt is consisted of (Goff et al., 1991). Phosphates, for example, have a weak acidifying activity as they are absorbed only slightly better than the corresponding cations (Horst et al., 1997). NaCl is neutral salt as both elements are absorbed completely and none is metabolized, so it does not affect neither DCAD nor acid-base status.


1magnesium sulfate heptahydrate (Epsom salt), 2magnesium chloride hexahydrate, 3calcium sulfate dihydrate (gypsum), 4calcium chloride dihydrate, 5ammonium sulfate, 6amonium chloride, Mr – relative molecular weight of a substance (sum of atomic weights of all atoms in the molecule), Val – valence, g/Eq – equivalent weight (Mr/valence of cation), DCAD = (Na+K) – (Cl+S).

**Table 3.** Characteristics and chemical composition of anionic salts

Anionic salts are not harmless substances and require caution for the use and handling them. The main limiting factors are bitter and salty taste and potential toxicity of higher doses. NH4Cl is considered the most toxic, followed by CaCl2. The combination of salts is the best solution because it reduces the possibility of harmful effects (Oetzel, 2000). The most acceptable method of administration is mixing salts in the feed.

An alternative to the anionic salts is adding mineral acids directly into a diet. Very potent in causing systemic acidosis is hydrochloric acid (HCl), while sulfuric acid (H2SO4) is less efficient (Goff & Horst, 1997a). An additional advantage of mineral acids is that cows prefer diets with acidic taste rather than bitter-salty taste of anionic salts. Goff & Horst (1998) found that efficient prevention of MF can be achieved with only 1,5 Eq of HCl in the diet, and that with no adverse consequences up to 2,5 Eq of the acid can be fed daily. Moreover, the addition of HCl in the diet had, unlike anionic salts, a beneficial effect on the consumption of feed. For safety, it is not advisable to use pure acid or keep it on farm, but rather as commercial products in the form of acidified feeds where the acid is mixed with some feed as a carrier. Byproducts of fermentation are usually used for this purpose, and also soybean meal or sugar beet pulp (Goff & Horst, 1997a, 1998).

Feeding anionic diet few weeks before calving significantly reduces the incidences of MF and subclinical hypocalcaemia in the herd, and improves milk production and reproductive performances of cows in the subsequent lactation (Beede et al., 1991; Block, 1984; DeGroot et al., 2010). Metabolic acidosis caused by anionic diet is subclinical, mild and compensated, and with no significant impact on animal health, but may influence some physiological functions if lasts long enough. Increased sensitivity of PTH receptors in renal tissue contributes to increased production of 1,25-(OH)2D before parturition (Gaynor et al., 1989; Goff et al., 1991). Cows fed anionic diet have a higher content of 1,25-(OH)2D in plasma, although changes in secretion of PTH does not occur (Phillippo et al., 1994). The active form of vitamin D takes part in osteolysis with PTH (Horst et al., 1994; Horst, 1986) and stimulates Ca reabsorption in renal tubules (Goff, 1992). Since 1,25-(OH)2D is important for the functioning of both Ca compensation mechanisms, change in its secretion is considered as an important effect of anionic diet.

Oetzel (1993) recommended the application of anionic salts in the herds with high incidence of MF (>10%) if not possible to formulate low Ca diet, or to improve production and reproduction in the herd which apparently does not have high incidence of MF. Adding anions in the diet is, therefore, to be considered not only for preventing MF, but also for prevention of subclinical hypocalcaemia which is responsible for the frequent occurrence of other metabolic and reproductive disorders in puerperium (Horst et al., 1994). Beede (1992) states that even in well-kept herds, where cows are in proper condition with no major problems with metabolic disorders, an additional 250-500 kg of milk can be achieved by using anionic salts in diets before parturition probably because lower incidence of subclinical hypocalcaemia.

52 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

**Table 3.** Characteristics and chemical composition of anionic salts

acceptable method of administration is mixing salts in the feed.

also soybean meal or sugar beet pulp (Goff & Horst, 1997a, 1998).

as an important effect of anionic diet.

cation), DCAD = (Na+K) – (Cl+S).

Salt Mr Val g/Eq DM, % % of DM DCAD

MgSO4x7H2O 1 246,51 2 123,26 48,83 20,20 26,63 -16.613 MgCl2x6H2O 2 203,33 2 101,67 46,83 25,53 74,46 -21.002 CaSO4x2H2O 3 172,18 2 86,09 79,07 29,44 23,55 -14.691 CaCl2x2H2O 4 147,02 2 73,51 75,49 36,11 63,88 -18.018 (NH4)2SO4 5 132,14 2 66,07 100,0 21,20 24,26 -15.134 NH4Cl 6 53,49 1 53,49 100,0 26,19 66,27 -18.692 1magnesium sulfate heptahydrate (Epsom salt), 2magnesium chloride hexahydrate, 3calcium sulfate dihydrate (gypsum), 4calcium chloride dihydrate, 5ammonium sulfate, 6amonium chloride, Mr – relative molecular weight of a substance (sum of atomic weights of all atoms in the molecule), Val – valence, g/Eq – equivalent weight (Mr/valence of

Anionic salts are not harmless substances and require caution for the use and handling them. The main limiting factors are bitter and salty taste and potential toxicity of higher doses. NH4Cl is considered the most toxic, followed by CaCl2. The combination of salts is the best solution because it reduces the possibility of harmful effects (Oetzel, 2000). The most

An alternative to the anionic salts is adding mineral acids directly into a diet. Very potent in causing systemic acidosis is hydrochloric acid (HCl), while sulfuric acid (H2SO4) is less efficient (Goff & Horst, 1997a). An additional advantage of mineral acids is that cows prefer diets with acidic taste rather than bitter-salty taste of anionic salts. Goff & Horst (1998) found that efficient prevention of MF can be achieved with only 1,5 Eq of HCl in the diet, and that with no adverse consequences up to 2,5 Eq of the acid can be fed daily. Moreover, the addition of HCl in the diet had, unlike anionic salts, a beneficial effect on the consumption of feed. For safety, it is not advisable to use pure acid or keep it on farm, but rather as commercial products in the form of acidified feeds where the acid is mixed with some feed as a carrier. Byproducts of fermentation are usually used for this purpose, and

Feeding anionic diet few weeks before calving significantly reduces the incidences of MF and subclinical hypocalcaemia in the herd, and improves milk production and reproductive performances of cows in the subsequent lactation (Beede et al., 1991; Block, 1984; DeGroot et al., 2010). Metabolic acidosis caused by anionic diet is subclinical, mild and compensated, and with no significant impact on animal health, but may influence some physiological functions if lasts long enough. Increased sensitivity of PTH receptors in renal tissue contributes to increased production of 1,25-(OH)2D before parturition (Gaynor et al., 1989; Goff et al., 1991). Cows fed anionic diet have a higher content of 1,25-(OH)2D in plasma, although changes in secretion of PTH does not occur (Phillippo et al., 1994). The active form of vitamin D takes part in osteolysis with PTH (Horst et al., 1994; Horst, 1986) and stimulates Ca reabsorption in renal tubules (Goff, 1992). Since 1,25-(OH)2D is important for the functioning of both Ca compensation mechanisms, change in its secretion is considered

Ca Mg N Cl S mEq/kg DM

Based on results of earlier studies, Horst et al. (1997) found that the most effective diets for prevention of MF are those with DCAD from -50 to -100 mEq/kg DM, while others state that it is between -50 and -150 (Beede, 1995), or -200 (Horst et al., 1994). However, Beede et al. (1991) achieved a good preventive effect with DCAD -250 mEq/kg, and Goff et al. (1991) with -230 mEq/kg. Too low DCAD, irrespective of the amount of added salts, can reduce feed consumption because too strong metabolic acidosis which depresses the appetite in animals (Goff & Horst, 1997b). Preventive effect of anionic diet on MF incidence occurs only if DCAD is below -40 mEq/kg DM in most cases, regardless of the type and quantity of added salts.

A risk for many health and reproductive disorders in puerperium is increased in cows with hypocalcaemia (Curtis et al., 1985; Massey et al., 1993). The prevention of hypocalcaemia can, in addition to better health status of cows in puerperium, improve milk production and increase reproduction performances in the herd (Beede et al., 1991; Block, 1984; DeGroot et al., 2010). Comparing the milk production in healthy cows with those who suffered MF, Block (1984) found greater milk production by about 14% in healthy animals. The same author found that cows fed anionic diet in the dry period produced on average 486 kg (7,3%) more milk than those fed cationic diet. Beede et al. (1991) conducted a field experiment with 510 cows and showed three positive effects of anionic diet: enhanced Ca metabolism in periparturient period, increased milk production in subsequent lactation, and better reproductive performances. Cows fed anionic diet before parturition produced 327 kg more milk (about 3,6%) than control cows.

Because of unpleasant taste, anionic salts can have depressing effect on feed intake (Joyce et al., 1997; Vagnoni & Oetzel, 1998), but this is minimal if salts are properly used (Block, 1984; Moore et al., 2000; Oetzel et al., 1988). Up to 300 mEq of anions/kg DM (3-3,5 Eq of salts per day per cow) can be added to the diet without depressing effect on feed intake (Horst et al., 1994; Oetzel, 2000). Intake also could be influenced by the type of salt because more acidic salts have less favorable taste. Feed intake depression was at least expressed with MgSO4, and much more with other salts, mainly CaCl2 and NH4Cl (Oetzel & Barmore, 1993). Last two also have much higher acidifying activity in relation to MgSO4 .

The best and easiest method of application of anionic salts is mixing in TMR which effectively masks their taste. In the conventional feeding system application of anionic salts is difficult, but still possible. The salts should be manually mixed in silage or haylage, or in concentrates (Oetzel & Barmore, 1993). In the latter case, salts should be mixed in sufficient amount of concentrates containing palatable feeds, at least 2,3-2,6 kg (Oetzel & Barmore, 1993; Pehrson et al., 1999).

Oetzel (2000) finds that preventive effect of anionic salts can be achieved if cows consume anionic diet at least 5 days before calving, while others state that at least 10 days is necessary. Since the expected and actual date of calving are matter of discrepancies in practice, it is advisable to start feeding salts 3-4 weeks before expected parturition, at least 2-3 weeks, to ensure that most of the cows consume the diet at least 10 days (Beede 1992, 1995).

NRC (2001) gives fairly broad recommendations for the content of Ca in anionic diet for dry cows, from 0,6 to 1,5% DM, which is not difficult to formulate when anionic Ca salts are used. Anionic salts should not be fed if Ca intake is below 50 g/day (Oetzel, 1993). As hypomagnesaemia negatively affects Ca metabolism at the time of calving, many authors emphasize the importance of adequate content of Mg in dry cow diet (Sansom et al., 1983; Thilsing-Hansen et al., 2002). Recommendation has increased from earlier 0,20-0,25% to the current 0,35-0,40% DM either in standard or in anionic diet (NRC, 2001). Increasing dietary Mg up to 0,40% DM has no negative consequences so, even if not prove useful, poses no risk or any practical problem in the formulation of a diet when anionic Mg salts are used (Wang & Beede, 1992). On the other hand, the Mg level of 0,4% DM has been also set as the maximum tolerable (NRC, 2001).

If we assume that DCAD in efficient anionic diet should be around -100 mEq/kg DM, it means that the initial DCAD should not be greater than +200 to +250 (Horst et al., 1997). Therefore, the diet must be formulated using feeds with lower content of K, such as corn silage, grass hay, etc. If necessary, part of the forage with high content of K can be replaced with concentrates rich in fiber, such as brewers' grains, sugar beet pulp, malt sprouts, etc. The most common problem is grass silage and alfalfa silage because they can contain more than 3% K. Most cereal grains have almost neutral DCAD, while it is more negative only in rapeseed meal and brewers grains primarily due to high content of S (NRC, 2001).

In field conditions, a marked decrease in feed intake may occur when anionic salts are included in the diet for the first time, therefore, adjusting cows should last at least three days (Oetzel, 2000)

The most accurate biological indicator of the degree of acidification of the body is pH of urine (Vagnoni & Oetzel, 1998). Urinary pH in cows was decreased linearly with decreasing DCAD (Charbonneau et al., 2006; Ganjkhanlou et al., 2010). The optimum pH of urine to prevent puerperal hypocalcaemia in Holsteins fed anionic diet is 6 - 6,5 (Horst & Goff, 1997) or wider, 6 - 7 (Moore et al., 2000). In Jersey cows, however, the pH of urine is necessary to reduce to 5,8 - 6,2. If the pH is lower than the specified, amount of anionic salts should be reduced and vice versa.

#### *4.3.2. Low calcium diet*

54 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

1993; Pehrson et al., 1999).

maximum tolerable (NRC, 2001).

days (Oetzel, 2000)

reduced and vice versa.

The best and easiest method of application of anionic salts is mixing in TMR which effectively masks their taste. In the conventional feeding system application of anionic salts is difficult, but still possible. The salts should be manually mixed in silage or haylage, or in concentrates (Oetzel & Barmore, 1993). In the latter case, salts should be mixed in sufficient amount of concentrates containing palatable feeds, at least 2,3-2,6 kg (Oetzel & Barmore,

Oetzel (2000) finds that preventive effect of anionic salts can be achieved if cows consume anionic diet at least 5 days before calving, while others state that at least 10 days is necessary. Since the expected and actual date of calving are matter of discrepancies in practice, it is advisable to start feeding salts 3-4 weeks before expected parturition, at least 2-3 weeks, to

NRC (2001) gives fairly broad recommendations for the content of Ca in anionic diet for dry cows, from 0,6 to 1,5% DM, which is not difficult to formulate when anionic Ca salts are used. Anionic salts should not be fed if Ca intake is below 50 g/day (Oetzel, 1993). As hypomagnesaemia negatively affects Ca metabolism at the time of calving, many authors emphasize the importance of adequate content of Mg in dry cow diet (Sansom et al., 1983; Thilsing-Hansen et al., 2002). Recommendation has increased from earlier 0,20-0,25% to the current 0,35-0,40% DM either in standard or in anionic diet (NRC, 2001). Increasing dietary Mg up to 0,40% DM has no negative consequences so, even if not prove useful, poses no risk or any practical problem in the formulation of a diet when anionic Mg salts are used (Wang & Beede, 1992). On the other hand, the Mg level of 0,4% DM has been also set as the

If we assume that DCAD in efficient anionic diet should be around -100 mEq/kg DM, it means that the initial DCAD should not be greater than +200 to +250 (Horst et al., 1997). Therefore, the diet must be formulated using feeds with lower content of K, such as corn silage, grass hay, etc. If necessary, part of the forage with high content of K can be replaced with concentrates rich in fiber, such as brewers' grains, sugar beet pulp, malt sprouts, etc. The most common problem is grass silage and alfalfa silage because they can contain more than 3% K. Most cereal grains have almost neutral DCAD, while it is more negative only in

In field conditions, a marked decrease in feed intake may occur when anionic salts are included in the diet for the first time, therefore, adjusting cows should last at least three

The most accurate biological indicator of the degree of acidification of the body is pH of urine (Vagnoni & Oetzel, 1998). Urinary pH in cows was decreased linearly with decreasing DCAD (Charbonneau et al., 2006; Ganjkhanlou et al., 2010). The optimum pH of urine to prevent puerperal hypocalcaemia in Holsteins fed anionic diet is 6 - 6,5 (Horst & Goff, 1997) or wider, 6 - 7 (Moore et al., 2000). In Jersey cows, however, the pH of urine is necessary to reduce to 5,8 - 6,2. If the pH is lower than the specified, amount of anionic salts should be

rapeseed meal and brewers grains primarily due to high content of S (NRC, 2001).

ensure that most of the cows consume the diet at least 10 days (Beede 1992, 1995).

Maintenance requirements of the cow and requirements of fetus during high pregnancy are usually satisfied with 35-45 g Ca/day (NRC, 2001). The content of Ca in the diet is often beyond that, and the requirements in this case are met mainly by passive absorption, while mechanisms of mobilization of body reserves and active absorption of Ca in the intestine are suppressed. Inactivity of these mechanisms in the dry period makes cows difficult to adapt to the sudden loss of Ca in colostrum at the moment of mammary gland activation. A few days required for starting these mechanisms causes temporary crisis in Ca homeostasis that results in decline of Ca in the blood (Goff, 1992).

When Ca content in the diet is below the minimum requirement, the animal is dependent on Ca mobilization from bone with simultaneous increase of active Ca absorption in the gut (Horst, 1986). These mechanisms can be activated before parturition and maintained active by constant stimulation up to the critical moment of parturition. Restriction of Ca intake to below 20 g/day caused negative balance of Ca and stimulates the secretion of PTH which increases tubular Ca reabsorption, bone resorption and production of 1,25-(OH)2D (Goff, 1992; Horst et al., 1997). This allows the cow to use Ca more efficiently from the diet and from body reserves immediately after calving (Goff, 1992). Introduction of low Ca diet leads to a slight decrease of Ca and P concentrations in plasma which returns to the base level in 3-4 days due to simultaneous increase in secretion of PTH that remained increased up to calving time (Goings et al., 1974). Prolonged exposure of the tissues to elevated PTH levels after feeding low Ca diet can overcome tissue resistance to PTH that might be induced by high dietary K (Goff, 2006a). This is the way to reduce or avoid period of adaptation to sudden losses of Ca which usually takes several days (Goff, 1992). Rations formulated to contain less than 20 grams of Ca proved to be very effective in preventing MF and significantly reduce the occurrence of hypocalcaemia too (Goings et al., 1971, 1974; Wiggers et al., 1975). For the best effect, restrictions of Ca consumption in the dry period should last at last 7-10 days (Goings et al., 1974; Wiggers et al., 1975) and be folowed by a high Ca diet immediately after calving (Horst et al., 1994; Oetzel, 2000). Cows fed in this way at the moment of calving mobilize about 10 g of Ca from the skeleton daily that is enough to prevent the occurrence of MF (Wiggers et al., 1975). Increasing Ca in the diet above daily needs and manipulation of its content within these limits does not affect the blood Ca status if other dietary minerals are maintained unchanged (Goff & Horst, 1997b).

Feeding low Ca diet to dry cows is considered the traditional way of preventing MF. For this reason, the use of feeds rich in Ca is usually avoided and the diet is formulated with corn silage and grass hay. Consumption of Ca can be reduced to 50-60 g/day in this way what often gives good results in the field (Goff, 1992; Horst et al., 1997). However, significant stimulation of parathyroid gland and complete preventive effect can be achieved only if Ca consumption is restricted to below 20 g/day. The formulation of such diet is, unfortunately, unrealistic in practice and that is the reason for its limited application (Goff, 1992). This type of diet has no adverse effects on production in the subsequent lactation (Goings et al., 1974), but the problem is, in addition to impracticality, that it may not last longer than 2-3 weeks because negative balance of Ca can be too exhausting for body reserves (Van Saun & Sniffen, 1996).

In the strategy for prevention of MF, Horst et al. (1997) recommended measures depending on the baseline cation-anion difference of the diet (Fig. 1). If it is less than 250 mEq/kg DM, the use of chloride and sulfate is justified without danger of low feed intake. If this value is above 250 mEq/kg DM it is necessary to consider other preventive measures, such as low Ca diet, short-term administration of oral Ca salts or some pharmacologic therapy (vitamin D analogs and active metabolites, PTH injections, etc.).

**Figure 1.** Strategy for preventing milk fever (adapted from Horst et al., 1997)

Thilsing-Hansen et al. (2002) rewieved research conducted over the past 50 years and concluded that any measures for preventing MF, even if used under ideal conditions, rarely reach preventive effect of 100%. The same authors calculated that efficiency of low Ca diets is best, reaching 80-100%, and anionic diets efficiency was 65-80%.

## **4.4. Prevention of hypomagnesaemia**

Commonly used parameter to characterize the grass tetany potential of forage is the ratio [K/(Ca+Mg)] (Mayland, 1988). Forages containing less than 0,2% Mg and a "tetany ratio" [K/(Ca+Mg)] greater than 2,2 have increased risk of inducing grass tetany (Crawford et al., 1998). The fertilizers containing N and K are the most important factors increasing [K/(Ca+Mg)] ratio in forages. Key step in nutritional prevention of hypomagnesaemia is finding a forage with lower ratio [K/(Ca+Mg)], or modify this ratio in the diet by adding more Ca and Mg supplements. Commercial grade MgO, MgCl2, MgCO3 and MgSO4 are good sources. As hypomagnesaemia may induce hypocalcaemia, including Ca in supplements to prevent Mg tetany may have a beneficial effect (Robinson et al., 1989).

NRC (2001) recommends Mg level of 0,35-0,40% DM in the diet for dry cows, although studies showed no benefit from increasing dietary Mg above 0.2% DM when diets contain less than 3% K (Wang & Beede, 1992). Van Saun & Sniffen (1996) recommended increasing dietary Mg above 0,20% when the content of K exceeds 1,2 to 1,5% DM. However, Goff (2006a) recommends that Mg content of the close-up dry cow ration and the early lactation ration should be between 0,35 and 0,40% as insurance against the possibility that the active transport processes for Mg absorption are impaired. In pregnant cows high Ca intake decreased utilization of dietary Mg and increases its excretion in urine (Sansom et al., 1983) that can be even worsened in metabolic acidosis (Wang & Beede, 1992). So it seems logical to increase Mg content in anionic close-up diet because it usually contains more Ca.

## **4.5. Prevention of milk fat depression**

56 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

**Figure 1.** Strategy for preventing milk fever (adapted from Horst et al., 1997)

is best, reaching 80-100%, and anionic diets efficiency was 65-80%.

**4.4. Prevention of hypomagnesaemia** 

analogs and active metabolites, PTH injections, etc.).

In the strategy for prevention of MF, Horst et al. (1997) recommended measures depending on the baseline cation-anion difference of the diet (Fig. 1). If it is less than 250 mEq/kg DM, the use of chloride and sulfate is justified without danger of low feed intake. If this value is above 250 mEq/kg DM it is necessary to consider other preventive measures, such as low Ca diet, short-term administration of oral Ca salts or some pharmacologic therapy (vitamin D

Thilsing-Hansen et al. (2002) rewieved research conducted over the past 50 years and concluded that any measures for preventing MF, even if used under ideal conditions, rarely reach preventive effect of 100%. The same authors calculated that efficiency of low Ca diets

Commonly used parameter to characterize the grass tetany potential of forage is the ratio [K/(Ca+Mg)] (Mayland, 1988). Forages containing less than 0,2% Mg and a "tetany ratio" [K/(Ca+Mg)] greater than 2,2 have increased risk of inducing grass tetany (Crawford et al., 1998). The fertilizers containing N and K are the most important factors increasing [K/(Ca+Mg)] ratio in forages. Key step in nutritional prevention of hypomagnesaemia is finding a forage with lower ratio [K/(Ca+Mg)], or modify this ratio in the diet by adding more Ca and Mg supplements. Commercial grade MgO, MgCl2, MgCO3 and MgSO4 are good sources. As hypomagnesaemia may induce hypocalcaemia, including Ca in supplements to prevent Mg tetany may have a beneficial effect (Robinson et al., 1989).

NRC (2001) recommends Mg level of 0,35-0,40% DM in the diet for dry cows, although studies showed no benefit from increasing dietary Mg above 0.2% DM when diets contain less than 3% K (Wang & Beede, 1992). Van Saun & Sniffen (1996) recommended increasing dietary Mg above 0,20% when the content of K exceeds 1,2 to 1,5% DM. However, Goff (2006a) recommends that Mg content of the close-up dry cow ration and the early lactation ration should be between 0,35 and 0,40% as insurance against the possibility that the active transport processes for Mg absorption are impaired. In pregnant cows high Ca intake decreased utilization of dietary Mg and increases its excretion in urine (Sansom et al., 1983) As milk fat depression has been observed over a wide range of feeding situations, this problem on dairy farms remains one of the more challenging tasks within overall nutritional management of dairy cows. Diets high in concentrates and low in fiber, and diets supplemented with plant or fish oil are the most often associated with MFD, but many other dietary factors also can affect milk fat synthesis including those which are able to alter rumen environment and those related to supply of polyunsaturated fatty acids (PUFA). Factors that can alter rumen environment include low level of physically effective NDF, feed particle size, total fiber in the diet, starch and non-structural carbohydrates, feeding pattern etc. On the other side, factors related to supply of PUFA are variation in fat content and fatty acid composition of feed ingredients, amount and availability of PUFA, and also feeding pattern.

Factor that alter rumen environment are first to consider in nutritional strategy to prevent or solve the problem of MFD, and those who affect rumen pH are the most important. Lower then normal ruminal pH, even without signs of acidosis, causes the change in bacterial population favoring those that have alternative pathways of biohydrogenation of dietary fatty acids. Inclusion of adequate level of so-called ''effective fiber'' and appropriate buffers in lactation diets can prevent drop in ruminal pH and markedly decrease MFD (Bergen, 2009). Forages, including long stemmed hay, are the main sources of effective fiber, thus the best method to maintain an adequate fat percentage in milk is to feed a balanced ration with adequate forage. The low levels of effective fiber may result from overfeeding of concentrates or the lack of forage, from consumption of large amounts of lush pasture and from silage or haylage that is too finely chopped (Perfield & Bauman, 2005).

Additional management practices to maintain a stable milk fat percentage in dairy herds include regular feeding of diet without abrupt changes, as well as feeding buffers such as Na-bicarbonate and/or Mg-oxide. Buffers are particularly useful when more than 5,5 kg of concentrate is fed per feeding and when frequent changes in diet are made.

Adding different sources of fat in dairy cow rations is a practice which has been favored when it is necessary to increase the energy consumption. However, higher amounts of fat can inhibit the activity of rumen bacteria and reduce the efficiency of fiber digestion, thus leading to a reduction in milk fat content (Schroeder et al., 2004). Although unsaturated vegetable oils can have a number of positive effects on fatty acid composition of milk fat, a negative effect on fat content must not be neglected due to the fact that the fat in milk is one of the main determinants of the price of milk and profits that farmers make. High levels of unsaturated oil in the diet can reduce fat content in milk along with possibly other adverse effects on production performances, such as drop in milk yield, decreased protein content in milk, low feed intake, etc. (Shingfield et al., 2006). Loor et al. (2002) found that the upper limit for dietary supplementation of unsaturated vegetable oils is 3,5% DM without serious nefative consequences on production. If fish oil is the supplement, it is at most 1% DM (Donovan et al., 2000).

## **Author details**

Cazim Crnkic and Aida Hodzic *University of Sarajevo, Veterinary faculty, Bosnia & Herzegovina* 

## **5. References**


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