THESIS

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thesis entitled

VARYING CATION-ANION DIFFERENCE
IN DIETS OF PREPARTUM DAIRY COWS

presented by

Stanley Joseph Moore

has been accepted towards fulfillment
of the requirements for

M.S. degree in Animal Science

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WWI

VARYING CATION-ANION DIFFERENCE
IN DIETS OF PREPARTUM DAIRY COWS

By

Stanley Joseph Moore

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Animal Science

1997

ABSTRACT

VARIN G CATION-ANION DIFFERENCE
IN DIETS OF PREPARTUM DAIRY COWS

By

Stanley Joseph Moore

Holstein cows (n=27) and heifers (n=35) were fed a close-up dry cow diet with a
varying dietary cation-anion difference (DCAD) of +14.4 (control), 0, or -15 meq/ 100g
dietary DM. Dietary Ca concentration increased (with CaCO3 supplementation) with
decreasing DCAD. Cows were fed experimental diets for 24 (1 prior to expected calving
date. Urine pH decreased with decreasing DCAD. Plasma HCO3 was lower with the -15
DCAD treatment. Prepartum DMI was depressed with the -15 DCAD treatment
compared with 0 DCAD treatment. BW also was reduced at 1 wk prepartum in the -15
DCAD treatment. Energy status in the 2 wk prepartum period was lower for cows fed the
-15 DCAD diet. Plasma NEFA, IGF-I, and hydroxyproline concentrations were not
affected by treatment. Decreasing DCAD increased plasma iCa concentrations both
prepartum and at the time of calving in cows but not heifers. Control cows had higher PTH
and calcitriol concentrations than cows on the O and -15 meq/ 100 g DM. In conclusion,
feeding anionic salts plus CaCO3 to reduce DCAD to -15 and increase Ca in prepartum diets
prevented hypocalcemia at calving in cows, but also decreased prepartum feed intake and

energy balance. Heifers did not become hypocalcemic and should not be fed anionic salts.

ACKNOWLEDGEMENTS

This project involved the work of many dedicated people. I thank Dr. Herb
Bucholtz for serving as my major professor and for his direction throughout my M. S.
program. For serving as my guidance committee, I thank Dr. Mike Allen, Dr. Tom Herdt,
Dr. Dave Beede, and Dr. Mike VandeHaar. I also thank Dr. Roy Emery for his assistance at
the MSU farm. I thank Tom Pilbeam for all his advice and assistance throughout the
project. I also thank Jim Liesman for his help with statistical analysis and Dr. Bal Shanna
for laboratory expertise and for helping with farm labor supervision.

I wish to thank Michigan State University for the financial support of this
experiment through Michigan Animal Industry Initiative Research and Extension Funding.

I wish to thank my parents who started me on my educational journey and supported
me throughout. Finally, thank-you Gayle for all your encouragement, support, and

dedication in helping me see through this project.

iii

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. vi
LIST OF FIGURES .......................................................................................................... vii
INTRODUCTION ............................................................................................................... 1
LITERATURE REVIEW .................................................................................................... 4
Dietary Ca effects on Ca status of the peripartum cow .................................................... 4
PTH effects on Ca status of the peripartum cow ............................................................. 5
Calcitriol effects on Ca status of the peripartum cow ...................................................... 7
Acid-base status effects on Ca status of the peripartum cow ........................................... 9
Dietary cation-anion difference (DCAD) effects on Ca status of the peripartum
cow ............................................................................................................................ 10
MATERIALS AND METHODS ....................................................................................... 13
Cows ............................................................................................................................. 13
Timeline of experiment .................................................................................................. 13
Treatment diets ............................................................................................................... 14
Housing and management of cows ................................................................................ 17
Blood and urine sampling .............................................................................................. 17
Blood minerals and pH analyses .................................................................................... l8
NEF A analysis ............................................................................................................... l8
IGF-I analysis ................................................................................................................. 18
Energy balance ............................................................................................................... 19
Calcitriol, hydroxyproline, and PTH analyses ............................................................... 19
Statistics ......................................................................................................................... 20
RESULTS AND DISCUSSION ........................................................................................ 22
Plasma mineral elements ................................................................................................ 22
Plasma hydroxyproline, PTH, and calcitriol .................................................................. 24
Acid-base balance: urine pH, plasma pH, plasma pCOz, and plasma HCO3 ................ 31

iv

Dry matter intake ........................................................................................................... 33

Body condition score and body weight .......................................................................... 36
Energy balance ............................................................................................................... 36
NEFA concentrations in plasma .................................................................................... 37
Plasma IGF-I .................................................................................................................. 38
Calf weight ..................................................................................................................... 39
Milk yield and components ............................................................................................ 39
Incidence of metabolic disorders ................................................................................... 41
SUMMARY AND CONCLUSIONS ................................................................................ 43
APPENDIX ........................................................................................................................ 46
BIBLIOGRAPHY .............................................................................................................. SO

LIST OF TABLES

Table 1. Ingredient and chemical composition of prepartum diets .................................... 15
Table 2. Ingredient and chemical composition of lactation diet ........................................ 16

Table 3. Plasma mineral element concentrations (mg/d1) for treatments (first 2 wk on
treatments) ...................................................................................................................... 22

Table 4. Effects of treatments, irrespective of parity, on plasma mineral element
concentrations immediately after calving (Oh, 12h, and 1d) .......................................... 23

Table 5. Treatment effects on plasma hydroxyproline (ug/ml), PTH (pg/ml), and
calcitriol (pg/ml) concentrations .................................................................................... 26

Table 6. Effects of treatment on acid-base status of cows up to 1d prepartum .................. 32

Table 7. Effects of treatment on acid-base status of cows immediately after calving (Oh,
12h, and 1d) ................................................................................................................... 33

Table 8. Effect of parity on DMI (kg) from -3d to +2d on treatments ............................... 33

Table 9. Treatment effects on prepartum and postpartum DMI (kg/d), BW (kg),
and BCS ......................................................................................................................... 34

Table 10. Energy balance (Mcal/d) as affected by treatment, prepartum and postpartum 37

Table 11. Plasma NEF A (uM) and IGF-1 (ng/ml) concentrations by treatment ................ 37
Table 12. Treatment effects on calf weight (kg) ................................................................ 39
Table 13. Treatment effects on daily milk yield and components ..................................... 40
Table 14. Incidence of metabolic disorders ....................................................................... 42

vi

LIST OF FIGURES

Figure 1. Parity by treatment effect on plasma iCa concentrations (mg/d1) (P < 0.05)

SEM = 0.11 .................................................................................................................... 24
Figure 2. Parity by day effects on plasma hydroxyproline concentrations (ug/ml)

(P < 0.001) SEM = 3.45 ................................................................................................. 26
Figure 3. Treatment by day interaction on plasma PTH concentrations (pg/ml) ............... 28
Figure 4. Parity by day interaction on plasma PTH concentrations (pg/ml) ...................... 28

Figure 5. Treatment by day effects on plasma calcitriol concentrations (pg/ml) of
primiparous cows in the peripartum period ................................................................... 30

Figure 6. Treatment by day effects on plasma calcitriol concentrations (pg/ml) of

multiparous cows in the peripartum period ................................................................... 31
Figure 7. Effect of treatment by day interaction on DMI (P < 0.001) SEM = 0.69 ........... 34
Figure 8. Parity by week interaction on DMI (P < 0.001) SEM = .56 ............................... 35
Figure 9. Plasma NEFA concentrations (uM) before and after parturition (P < 0.001)

SEM = 74.2 .................................................................................................................... 38
Figure 10. Treatment by week interaction on 4% F CM (kg), (P < 0.05) SEM = .938 ...... 40

vii

INTRODUCTION

Milk fever costs the US. dairy industry over $120 million/yr in direct costs of
treatment and secondary problems (Goff and Horst, 1990). The dynamics of feeding and
managing high producing cows are most critical for the period 30 d prepartum through 30
d postpartum.

Besides tremendous changes in energy and protein flux around calving,
peripartum cows also experience large changes in mineral element dynamics. Daily body
turnover of Ca changes from about 10 g in dry cows to about 35 g in lactating cows
(Moodie, 1960). In a recent study, almost 70% of multiparous cows in three commercial
farms in Florida, Colorado and Wisconsin suffered from clinical or subclinical
hypocalcemia at calving, although only 8% exhibited clinical hypocalcemia (i.e., milk
fever) (Wang, 1992). Milk fever, also called parturient paresis, occurs when the output of
Ca from the blood exceeds the input of Ca absorbed from the gut and resorbed from bone
(Wang et al., 1994; Ward et al., 1953). It has been suggested that because Ca has a role
in smooth muscle function, hypocalcemia is either the root cause or a predisposing factor
for several other problems in the peripartum cow. Parturient paresis is a risk factor for
dystocia (7.5 t012.6 odds ratio), prolapsed uterus (13.1 to 34.6 odds ratio), retained
placenta (2.0 to 2.8 odds ratio), and early metritis (1.2 to 1.8 odds ratio) (Grohn et al.,

1990) (where odds ratio indicates the increased likelihood that a cow with parturient
1

2

paresis would develop one of these metabolic disorders or diseases, compared with cows
without parturient paresis).

Decreasing the dietary cation-anion difference (DCAD; meq [(N a + K) - (Cl +
S)/100g DM]) during the last 3 to 4 wk before calving can have beneficial effects on
systemic acid-base status, Ca metabolism, peripartum health, and postpartum productive
and reproductive performance (Beede, 1995; Beede, 1992; Horst et al., 1994; Oetzel et
al., 1988; Tucker et al. 1991).

The strategy of feeding anions (C1 or S) to lower the DCAD of prepartum diets is
often successful. These anions are often supplied by the so-called “anionic salts”
(MgSO4-7H20, NH4Cl, (NH4)ZSO4, CaClZOZHzO, MgClze6H20, and CaSO402H20).
However, the optimum DCAD, and thus the amount and kinds of anionic salts to feed are
not well established.

The common recommendation is to add anionic salts until the DCAD value is -10
to ~15 meq/100g DM. However, Michigan forages typically are high in K, thus requiring
large amounts of anionic salts to achieve a diet with a negative DCAD of -10 to -15
meq/ 100g DM. Based on experience in an earlier dry cow study at Michigan State
University (V andeHaar et al., 1995), protection against hypocalcemia may be achieved
with only moderate additions of anionic salts to dry cow diets. Most studies to date have
examined DCAD of -10 to -15 to prove that the DCAD concept was valid. However,
anionic salts are unpalatable and a moderate inclusion rate (to achieve DCAD = 0
meq/100g DM) may have less effect on feed intake than higher inclusion rate, and thus be

desirable to optimize energy, protein, and mineral nutrition simultaneously. If moderate

3
inclusion rates could be effective, prepartum diets could be formulated to help control

hypocalcemia yet maintain high DMI, thus reducing the incidences of ketosis and fatty
liver.

The hypothesis tested in this study was that 1) feeding a close-up dry cow diet
with a DCAD of 0 meq/100g DM would not prevent hypocalcemia in the peripartum
dairy cow to the same extent that a diet with a DCAD of -l 5 meq/100g DM would, and 2)
that feeding a diet with 0 meq/100g DM DCAD diet would not result in a higher DMI
compared with a diet of -15 meq/100g DM, and thus would not result in less lipid
mobilization before calving.

The alternative hypothesis was that the 0 meq/ 100 g DM DCAD would prevent
hypocalcemia and depression of DMI compared with the -15 meq/100g DM DCAD diet.

The objectives of the research were to examine the effects of a prepartum diet
containing no (Control, DCAD = +14.4 meq/100g DM), moderate (DCAD = 0 meq/100g
DM) or high (DCAD = -15 meq/100g DM) amounts of anionic salts on the prevention of
parturient hypocalcemia, prepartum feed intake, and health and milk production
postpartum. By experimental design, the dietary Ca concentration varied among the three
treatments. The dietary Ca concentration increased (with supplemental CaCO3) with

increased inclusion of the anionic salts (decreasing DCAD).

LITERATURE REVIEW

Dietary Ca effects on Ca status of the peripartum cow

Past research on preventing parturient paresis and field recommendations have
focused on changing the Ca/P ratio or reducing the amount of Ca in the prepartum diet
(Boda and Cole, 1954; Verdaris and Evans, 1974). A later review of literature on Ca/P
ratio showed that Ca/P ratio had no significant effect on the Ca status of the cow (Beitz et
al., 1973; Jonsson, 1978).

Reducing the amount of dietary Ca to prevent parturient paresis is considered to
be impractical for most dairy farms (Goff et al., 1988). Very low Ca diets (<20 g/d)
result in low blood Ca and thus increase parathyroid hormone (PTH) and calcitriol (1 ,25-
(OH)2-D3) before calving (Bushinsky et al., 1982; Goff, 1992a; Jonsson et al., 1980;
Kumar, 1980).

Jonsson (1978) showed that 37 g/d of dietary Ca was not low enough to prevent
milk fever. Similarly, Jonsson et a1. (1980), showed no effect of dietary Ca concentration
on milk fever at the inclusion rate of 37 to 150 g/d. Oetzel (1991) in a meta-analysis of
data from the literature showed that incidence of milk fever increased with increasing
dietary Ca up to about 1.2% of the diet, and beyond 1.2% milk fever incidence actually
declined. Verdaris and Evans (1976) showed that with very high amounts of dietary Ca

in the prepartum period (2.1% of DM), the amounts of Ca absorbed and retained were
4

5

increased. This increased absorption can be very important in older cows because
reduced Ca digestibility and absorption cause older cows to be at increased risk for milk
fever (Hansard et al., 1954; Moodie, 1960). Moodie (1960) suggested that decreased gut
motility caused the decrease in Ca absorption. The major routes for Ca homeostatic
control are absorption, bone tissue deposition and bone resorption, urinary loss, and
resorption from kidney tubules (Miller, 1974). Parathyroid hormone, calcitriol, and acid-
base status are some of the major controlling factors of Ca absorption, and bone tissue

deposition and resorption in the cow.

PTH effects on Ca status of the peripartum cow

Parathyroid hormone (PTH) is secreted by the parathyroid gland in response to
low blood Ca (Boda and Cole, 1954; Kumar, 1980; Ramberg et al., 1967) and PTH
concentration is inversely proportional to the blood Ca concentration. PTH increases
distal tubular reabsorption of Ca in the kidneys (Ganong, 1985), increases bone Ca
resorption (Block, 1992; Boda and Cole, 1954), and mediates an increase of calcitriol in
blood (Kumar, 1980; Wang et al., 1994).

PTH affects bone by causing a increase in Ca permeability in osteoblasts,
osteocytes, and osteoclasts. The osteoclasts erode and resorb previously formed bone Ca
(Block, 1992; Ganong, 1985). Bone is composed of both labile (available) and stable
(unavailable) forms of Ca (Moodie, 1960). PTH first acts on available bone Ca stores,
but will eventually affect even the stable portion of bone Ca (Goff, 1992b). PTH effects
on bone can be measured by looking at hydroxyproline concentrations in blood (Leclerc

and Block, 1989; Wang et al., 1994). Increased blood hydroxyproline indicates increased

6

resorption of bone. The Ca that is released from bone due to the action of PTH, is
released as Ca phosphate (Barzel and Jowsey, 1969) and dissociates in the blood to form
Ca and P046.

PTH affects calcitriol concentrations in the blood by activating renal
mitochondrial l-alpha-hydroxylase which converts 25-OH-D3 to calcitriol (Goff, 1992b;
Kumar, 1980; Wang et al., 1994). Ikeda et a1. (1987) showed that in rats changes in renal
l-alpha-hydroxylase activity generally paralleled those of circulating calcitriol.

PTH is regulated through several mechanisms. As mentioned earlier, PTH is
correlated negatively with blood Ca concentration (Goff, 1992a). Ramberg et a1. (1967)
reported that PTH secretion was inversely proportional to Ca concentration in plasma.
D’Amour et a1. (1986) showed that in calves, dogs and humans, hypercalcemia caused a
decrease in PTH concentration to 40% of basal concentrations. Hypocalcemia increased
PTH concentrations 300 to 450% above basal concentrations. Hughes and Haussler
(1978) showed that the parathyroid gland has receptors for calcitriol. This may serve as
feedback regulation to shut down PTH secretion after calcitriol is produced and low
blood Ca is corrected.

Lastly, the effect of PTH on bone tissue and renal production of calcitriol is
dependent in part on the acid-base balance of the cow (Goff, 1992b). Metabolic acidosis
causes bone to be more sensitive to PTH stimulation, whereas metabolic alkalosis causes
bone and renal tissues to be refractory to PTH stimulation (Goff, 1992a). Block (1992)
showed that hypocalcemic cows had higher plasma PTH concentrations. This would
suggest that low blood Ca is not due to a lack of PTH secretion, but instead a lack of bone

and renal tissue sensitivity to the effects of PTH.

Calcitriol effects on Ca status of the peripartum cow

Calcitriol increases blood Ca status by increasing uptake from the intestine
(Block, 1992; Evans, 1977; Gaynor et al., 1989; Goff, 1992a). This uptake is by active
transport (transcellular) across the intestinal epithelium (Goff, 1992b). Calcitriol also
enhances mobilization of Ca from bone (Kumar, 1980; Wang etal., 1994). These
influences of calcitriol on Ca utilization have led to experimental use of calcitriol in the
prevention of milk fever. Given the correct timing and dose, calcitriol reduces incidence
of milk fever when given intramuscularly, intravenously, or orally (Boda, 1954; Hibbs
and Pounden, 1956; Littledike and Horst, 1982; Wang et al., 1994). However,
administration of calcitriol is not used widely for the prevention of milk fever due to the
need for exact timing of administration with respect to actual and potential toxicity.
Hibbs and Pounden (1956) showed that feeding 30 million units of calcitriol for 3 to 8 d
prepartum would prevent milk fever. When calcitriol was fed longer than 2 to 4 wk, the
positive results disappeared, presumably because the increased concentration of blood Ca
for too long caused the parathyroid gland to reduce secretion of PTH. Hove and
Kristiansen (1982) also found positive results with oral calcitriol. In their experiment, the
dose of calcitriol was 500 ug given 1 to 3 d prepartum. In 1980, Reinhardt and Conrad
found that giving calcitriol intravenously for 7 d prepartum initially raised circulating
concentrations of calcitriol in blood, but caused decreased circulating calcitriol
concentrations just prior to calving. These authors suggested that feedback inhibition was
overridden initially, but that the inhibition still occurred before parturition. Toxicity

generally occurs when calcitriol is given for an extended period of time. This toxicity

8
may cause calcification of internal organs (Wang et al., 1994) and eventually lead to

death. Littledike and Horst (1982) showed that a parenteral dose of calcitriol given 32 d
prepartum at 15 to 17.5 X 106 IU prevented milk fever, but it also caused vitamin D
toxicity and 10 of 17 cows died. Another concern with using calcitriol in prevention of
milk fever is cows becoming dependent on calcitriol administration. Calcitriol stimulates
Ca absorption from the intestine, making cows more dependent on Ca absorption to
maintain plasma concentrations and homeostasis as opposed to activating bone resorption
(Wang et al., 1994). Wang et al. (1994) also suggested that because vitamin D
metabolites inhibit l-alpha-hydroxylase in the kidney, there is an increasing dependency
on exogenous vitamin D sources.

Production of calcitriol from 25-(OH)-D3 by the kidneys is affected negatively by
dietary Ca intake (Bushinsky et al., 1982; Goff, 1992a; Ikeda et al., 1987; Kaetzel and
Scares, 1985; Kumar, 1980), and positively by increasing PTH blood concentrations
(Block, 1992; Jonsson, 1978). Calcitriol production is affected negatively by plasma Ca
concentrations (Block, 1992; Bushinsky et al., 1982; Goff, 1992a; Ikeda et al., 1987;
Jonsson, 1978), negatively by plasma P concentrations (Goff, 1992a; Goff, 1992b;
Kumar, 1980), and negatively by feedback inhibition of calcitriol (Gordeladze et al.,
1986; Jonsson, 1978; Reinhardt and Conrad, 1980; Wang et al., 1994). Lastly, calcitriol
production by the kidneys is affected negatively by a positive DCAD (Block, 1992;

Gaynor, 1989; Goff, 1992a).

9
Acid-base status effects on Ca status of the peripartum cow

Buffer systems are important in the cow as in other animals to maintain body
intracellular and extracellular pH. Two of the important buffer systems are: 1) carbon
dioxide/bicarbonate system in blood (Freeden, 1993), and 2) bone buffer system
(Bushinsky and Lechleider, 1987; Freeden, 1993). Maintaining a narrow range in pH in
extracellular and intracellular fluids is critical for enzyme systems which are pH-
dependent (Block, 1992).

The carbon dioxide/bicarbonate system is the main system for maintaining
acid/base status and is regulated by the kidneys and lungs. The concentration of
bicarbonate is determined by the difference in the concentrations of all cations and all
anions (excluding HCO3') in plasma (Fredeen, 1993). When cations exceed anions in the
blood, the kidney increases the excretion of bicarbonate into urine thus maintaining pH
(Block, 1992). When anions exceed cations in blood, the kidney will conserve HCO,‘ in
the plasma to maintain pH. Respiratory rate controls the plasma pC02 concentrations and
thus the concentration of carbonic acid (H2C03) in the blood (Fredeen, 1993). When
metabolic acidosis occurs carbonic acid increases in the blood as bicarbonate is converted
to carbonic acid. The carbonic acid is picked up by the red blood cells and transported to
the lungs for respiratory removal of CO2 (F redeen, 1993). This metabolic acidosis may
or may not include a decrease in plasma pH. If an increase in respiration rate does not
occur, a decrease in plasma pH will occur causing metabolic acidemia.

Bone contains a vast reserve of base (carbonate) (Fredeen, 1993). During acidosis
the solubility of Ca phosphate in bone (as hydroxyapatite) is increased (Barzel and

Jowsey, 1969; Block, 1992), thus increasing the entrance rate of Ca from bone into blood.

10

Ca phosphate dissociates in blood to form Ca and P044. Clearance rate of Ca also
increases with acidosis as excess Ca in blood is excreted in the urine. Metabolic acidosis
thus causes calciuria (increased Ca in the urine) (Fredeen, 1993; Block, 1992; Fredeen et
al., 1988). Although metabolic acidosis increases the flux through the exchangeable Ca
pool, it does not affect the size of the exchangeable Ca pool (Fredeen et al., 1988).
Because of these buffer systems, the Ca status of the cow can be affected by
manipulating the acid-base status of the cow. Any manipulation causing acidosis will
cause bone and renal tissues to be more sensitive to PTH stimulation (Goff, 1992a), and
cause the bone buffer system to release Ca phosphate (Barzel and Jowsey, 1969; Block,
1992). Manipulation of the acid-base status of the cow can be achieved through changing

the dietary cation-anion difference (Beede and Sanchez, 1989).

Dietary cation-anion difference (DCAD) effects on Ca status of the peripartum cow
Decreasing the DCAD of the diet during the last 3 to 4 wk prepartum affects
systemic acid-base status and has beneficial effects on Ca metabolism, peripartum health,
and postpartum productive and reproductive performance (Beede, 1995; Beede, 1992;
Horst et al., 1994; Oetzel et al., 1988; Tucker et al., 1991). DCAD is calculated for the
diet by subtracting the milliequivalents of anions from the milliequivalents of cations in
the ration. Rather than consider all cations and anions, the commonly used formula is
meq [(K + Na) - (C1 + S)]/100g dietary DM (Beede, 1995). Of these minerals elements,
K, Na and C1 are considered fixed ions because they are not metabolized. Sulfur,
although not a fixed ion, is included in the equation because S directly acidifies biological

fluids and can alter acid-base balance if included at high dietary concentrations (Block,

11
1992; Lemann and Rehnan, 1959; Tucker et al., 1991). The anionic salts used in the

present study (MgSO407HZO, CaC1202H20, CaSO402HzO) change acid-base status
because Cl and S have higher rates of absorption compared to Ca and Mg. The anions, C1
and S, then draw H+ ions out of base creating metabolic acidosis (Beede and Sanchez,
1989)

Decreasing DCAD in the prepartum period leads to increased plasma Ca
concentrations at parturition (Beede, 1995; Gaynor et al., 1989; Leclerc and Block, 1989;
Oetzel et al., 1988). This relationship between DCAD and plasma Ca concentration was
first reported by researchers in Norway (Dishington, 1975; Ender et al., 1962). The mode
of action of negative DCAD to increase plasma Ca is not well understood. Block (1992)
suggested that increased excretion of Ca in the urine leads to an initial drop in plasma Ca
concentrations. This drop in Ca concentration would increase the release of PTH and
increase the formation of calcitriol by the kidneys. Both of these responses to the initial
decrease in blood Ca would lead to an eventual increase in blood plasma Ca
concentrations.

This suggestion of an initial drop in blood Ca concentrations is not supported by
others. F redeen et a1. (1988) suggested instead that the pool size of Ca is not affected by
acidosis, but the flux of Ca is increased. An increase in dietary Ca concentration is
recommended with decreased DCAD diets because of this increased flux (Beede, 1995;
Fredeen, 1993; Wang, 1994). The increase in Ca entering the pool is apparently mostly
from increased bone mobilization as evidenced by increases in hydroxyproline

concentrations in blood and decreased bone density (Leclerc and Block, 1989; Petito and

12

Evans, 1984). The increase in Ca flux fi'om bone is due to the bone buffering system
trying to compensate for the metabolic acidosis initiated through a negative DCAD diet.
There is also evidence that bone may become more sensitive to PTH when animals are
kept in a mild state of metabolic acidosis as compared with an alkalinic state (Goff,
1992a; Goff, 1992b; Ochs et al., 1964).

A negative DCAD also may cause an increase in intestinal absorption of Ca
(Leclerc and Block, 1989; Wang and Beede, 1992). This increase in absorption may be
through increases in plasma calcitriol (Block, 1992; Gaynor et al., 1989; Goff, 1992b),
and (or) an increase in tissue sensitivity to calcitriol in the acidotic state created by

feeding a negative DCAD diet (Block, 1992; Goff, 1992b).

MATERIALS AND METHODS

Cows

Sixty-two Holstein cows (27 multiparous and 35 primiparous) from the Michigan

State University herd in East Lansing were selected for the experiment. Cows were

assigned randomly to one of three treatments and were assigned in blocks of three cows

each based on parity and date of expected calving. Only two cows were available for the

last block and they were assigned randomly to one of three treatments. Cows were

housed in individual tie-stalls to enable individual feeding and feed intake measurements.

Cows were brought into the experiment approximately 35 d prior to expected date of

calving on Monday each week and remained on the Control diet (DCAD = +14.4) for at

least 1 wk (see timeline) before being fed a treatment diet.

Timeline of experiment

 

Control Treatment Diets Lactation Diet

.Diet I I J
F r 1 I
-31 d -24 d 0 d 70 d
Pretreatment Treatment Calving

Period Period

13

1 4
Treatment diets

Twenty-four days prepartum, cows were assigned to one of three treatment diets.
The diets were 1) Control diet (DCAD: +14.4), 2) diet formulated for 0 DCAD, and 3)
diet formulated for -15 DCAD (Table 1). After calving all cows were placed on the same
lactation diet (Table 2). All diets were fed once daily with orts from the previous feeding
removed just prior to the next feeding. Amounts fed and orts removed were recorded to
calculate daily feed intakes. All forages were measured twice per week for DM% using a
Koster Tester (Koster Crop Tester, Inc., Strongsville, OH) by the farm crew.

Feed samples were collected once per week and analyzed via wet chemistry by
North East DHIA Forage Laboratory (NEDHIA) (Ithaca, NY) for mineral element
content and DM percent. Full analysis (NEL, CP, NDF, DM and mineral element

content) was done twice per month via wet chemistry through the same laboratory.

15

Table 1. Ingredient and chemical composition of prepartum diets.

 

 

Item Control 0 DCAD -15 DCAD
Dietary ingredients, % of dietary DM
Corn silage 34.9 34.9 34.9
Haylage 20.1 20.1 20.1
Corn, ground 26.0 24.0 22.0
Soybean meal 8.9 8.9 8.9
Pelleted cottonseed hulls 4.2 2.1 0.0
Trace mineral and vitamin premix 5.9 5.9 5.9
Anionic pack 0.0 3.9 7.9
Composition of anionic pack, % of dietary DM
Magnesium sulfate 0.0 0.24 0.50
Calcium sulfate 0.0 0.44 0.90
Calcium chloride 0.0 0.53 1.09
Limestone (CaCO3) 0.0 0.68 1.40
Dicalcium phosphate 0.0 0.15 0.30
Corn, ground 0.0 1.83 3.74
Calculated nutrient content, % of dietary DM
NEL (Mcal/kg) 1.66 1.65 1.63
CP 16.5 16.5 16.3
NDF 31.6 30.0 28.0
K 1.35 1.35 1.35
Na 0.12 0.12 0.12
Ca 0.44 0.97 1.50
P 0.34 0.37 0.39
Mg 0.20 0.24 0.28
Cl 0.44 0.70 0.96
S 0.21 0.32 0.44
DCAD, meq/100g DM +14.4 0 -15

 

16
Table 2. Ingredient and chemical composition of lactation diet.

 

 

Dietary ingredients % of DM
Corn Silage 17.3
Haylage 20.3
Corn, high moisture 17.6
Corn, ground 10.8
Soybean meal 15.6
Cottonseeds 7.8
Distillers grain 4.9
Blood meal 0.5
Trace minerals and vitamins 5.2
premix

Nutrient content

NEI (Mcal/kg) 1.70
CP 18.4
NDF 26.0
K 1.3

Na 0.36
Ca 1.07
P 0.50
Mg 0.3

DCAD, meq/100g of dietary DM +30.1

 

17

Housing and management of cows

Cows were milked three times per day at 0550, 1400, and 2200 h. Milk yield was
measured electronically (Boumatec, Madison, WI) and recorded daily using Dairy Comp
305 (Valley Agricultural Software, Tulare, CA). Milk samples were collected once a
week (from the three individual milkings for that day) and analyzed for fat, protein,
lactose, and SCC by Michigan DHIA (East Lansing, MI). Milk composition of fat,
protein, lactose, and SCC was calculated as the weighted average of all three milkings.

Body weights of all cows were measured weekly (Thursday and Friday) at 0900 h.
Body condition (1 to 5 scale) was assessed at the times of body weight measurement by
three individuals (Wildman, et al., 1982).

Calf birth weights were measured within 12 h of birth and recorded.

Blood and urine sampling

Blood samples were obtained from the tail vein twice per week (Monday and
Friday) at 1600 h, starting 1 wk afier cows entered the experiment. Two weeks prior to
expected date of calving, blood was sampled three times per week (Monday, Wednesday,
and Friday). Beginning 1 wk prior to expected date of calving, cows were bled daily until
calving. Cows also were bled after calving within 4 h, and at 12 h, 1 d, 2 d, 3 d, 1 wk,
and 2 wk. Lithium heparin vacutainer tubes were used for blood collection. Samples
were centrifuged and plasma collected for analysis. Samples were measured for
concentrations of iCa, K, Na, Cl, pH, pCOz, HCO3, calcitriol and PTH as indicators of the
Ca regulatory system, and non-esterfied fatty acids (N EPA) and insulin-like growth

factor-I (IGF-I) as indicators of energy status.

18
Urine pH was measured each Monday, Wednesday, and Friday before calving to

assess acid-base status. Hand-held urine pH meters (Hach, Loveland, CO) were

calibrated prior to each sampling using a pH 7.0 buffer solution as the standard.

Blood mineral elements and pH analyses

Blood mineral elements and pH analyses were done on plasma samples within 3 d
of sampling. Samples were stored at 4°C prior to analysis. Analyses for Na, K, Cl, iCa,
pH, pCOz, and HCO3 were done. Analysis was accomplished using a Nova Biomedical

Stat Profile 4, blood gas analyzer (Waltham, MA).

NEFA analysis
Samples were analyzed for NEF A using a NEFA-C kit (Wako Pure Chemicals
Inc., Richmond, VA) with modifications by Dyk (1995). Any plasma sample with a

coefficient of variation greater than 15% for two replications was reanalyzed.

IGF-I analysis
Plasma samples were analyzed for IGF-I using a radioirnmunoassay procedure (see

Appendix for procedure and all buffer and reagent mixtures) (Shanna et al., 1994).

1 9
Energy balance

Energy balance was calculated for each cow by week (-2, -1 wk prepartum and 2
through 9 wk postpartum) using the following equations:

Energy balance (Mcal/day)=(N E mm - NE mam - NE lactation)/eff1ciency factor.

NE intake = DMI(kg)"‘NEl

NE min, prepartum = BW(kg)'75*0.104 Mcal/kg'”

NE mm, postpartum = BW(kg)'75*0.08 Mcal/kg'"

NE ,mm = milk(kg)*(41 .63 *fat+24. 13 *prot+21 .60*1act-1 1.72)/1000
If energy balance was negative postpartum, the efficiency factor was 0.82 to account for
the efficiency of milk production from body tissue. An efficiency factor of l was used to
calculate energy status when status was positive postpartum and for prepartum energy

status.

Calcitriol, hydroxyproline, and PTH analyses

Calcitriol, hydroxyproline and PTH were analyzed by the USDA National Animal
Disease Center in Ames, Iowa under the direction of Dr. Jesse Goff. Plasma samples
were shipped frozen, on ice. Calcitriol was analyzed using a microassay procedure
developed by Reinhardt et al. (1984).

Dabev and Struck (1971) developed the hydroxyproline assay procedure. The
assay was modified for use in microtiter plates (Goff, personal communication).

PTH was analyzed with a kit from Nichols Diagnostics (San Juan Capistrano,

CA). The test used for PTH was an intact PTH immunoassay, which was a two-site

20

irnmunoradiometric assay for the measurement of the biologically intact 84 amino acid

chain of PTH. This human PTH kit has been validated for bovine (Goff et al., 1989).

Statistics

The GLM procedure was used in the analysis of all data using a repeat measure
design (SAS, 1995). All results were reported as least squares means. The model for
AN OVA included parity, block(parity), treatment, parity by treatment, treatment by
block(parity), time, treatment by time, parity by time, and parity by treatment by time for:
milk yield and components, DMI, body condition score, body weight, energy status, urine
pH, plasma mineral elements and pH, plasma pCOz, plasma HCO3, plasma IGF-I, and
plasma NEFA concentrations. “Time” in the model refers to week, or day depending on the
analysis. DNfl also was analyzed with the model including only parity, block(parity),
treatment, and parity by treatment due to inestimable contrasts when time was included.
Calf weights were analde with a model that included parity, block(parity), treatment, and
parity by treatment. Plasma hydroxyproline, PTH and calcitriol were analyzed with a
model that included parity, treatment, parity by treatment, cow(parity by treatment), time,
parity by time, treatment by day, and parity by treatment by day.

Due to heterogeneous variance in the PTH and calcitriol data, the log base 10 of the
data was used in the analysis.

For statistical analyses of blood plasma variables, pretreatment means of data for up

to 14 d on experimental diets were used as covariates.

21
Milk yield and component data were analyzed for 2 through 9 wk. Data from wk 1

and 10 were not included in the analysis due to missing data and varying time of sampling
with respect to calving.

Categorical data (e. g. as metabolic disorder incidences) were analyzed in SAS using
Fisher’s Exact Test (2-Tail). Data fi’om‘cows having twins were not included in the
analysis for metabolic disorder incidences, but were included in all other analyses.

Treatment means were compared by orthogonal contrasts: Control vs. 0 and -15

meq/100g dietary DM DCAD and 0 vs. -15 meq/100g dietary DM DCAD.

RESULTS AND DISCUSSION

Plasma mineral elements

Table 3 shows the plasma mineral element concentrations for the three treatments.
For blood samples taken during the first 2 wks on dietary treatments, iCa concentrations
were lower for Control than 0 and —l 5 DCAD treatments, but did not differ for 0 vs. —1 5
DCAD (P < 0.005, and P = 0.30 respectively). Concentration of K was lower for Control
than 0 and -15 DCAD (P < 0.001), but not different between 0 vs. -15 DCAD (P = 0.49).
Blood Na concentrations were not different due to treatment (Table 3). Concentration of
C1 in plasma was higher for primiparous than multiparous (385 vs. 379 mg/dl
respectively P < 0.05). Pooled across parities plasma Cl was lower for Control than 0 and

-15 DCAD, and lower for 0 than -15 DCAD (P < 0.05) (Table 3).

Table 3. Plasma mineral element concentrations (mg/d1) for treatments (first 2 wk on
treatments).

 

Item Control 0 DCAD -15 DCAD SEM C — 1 C — 2

 

iCa 4.65 4.77 4.82 0.038 P < .005 P = .30
K 16.90 17.38 17.49 0.11 P < .001 P = .49
Na 333 334 333 0.89 P = .32 P = .51
Cl 379 381 387 1.5 P < .05 P < .05

 

C - 1: +14 vs. 0 and -15 DCAD, C - 2:0 vs. -15 DCAD

22

23

Plasma mineral element concentrations (means of samples taken 0, 12 h, and 1 d
after calving), are shown in Table 4. There was an effect of treatment (P < 0.001), parity
(P < 0.001), and parity by treatment (P < 0.05) on plasma iCa concentrations (Table 4,
Figure 1). Cows on the Control diet had lower plasma iCa than those on 0 and -15
DCAD (P < 0.01), and plasma iCa was lower for 0 DCAD than for -15 DCAD (P < 0.05)
(iCa = 4.1, 4.2, and 4.5 mg/dl for Control, 0 and -15 DCAD, respectively). Figure 1
shows multiparous cows had lower plasma iCa concentrations than primiparous cows
(overall means across (1 = 4.0 and 4.5 mg/dl, respectively). This parity effect on Ca
concentration in blood is well documented (Barzel, 1969; Hansard et al., 1954; Moodie,
1960). Plasma iCa concentrations of primiparous cows were similar for all treatments
and indicated that primiparous cows were not hypocalcemic at parturition (i.e. iCa > 4
mg/dl) (Figure 1). However, plasma iCa concentration of multiparous cows increased
from 3.7 to 3.9 and 4.4 mg/dl for Control, 0, and -15 DCAD, respectively 12 h after
calving. Thus, multiparous cows fed the Control and 0 DCAD were hypocalcemic (iCa 5
4 mg/dl), whereas cows fed the -15 DCAD had mean plasma iCa concentrations well

above 4 mg/dl at all sampling times.

Table 4. Effects of treatments, irrespective of parity, on plasma mineral element
concentrations immediately after calving (0h, 12h, and 1d)

 

Item Control 0 DCAD -15 DCAD SEM C - 1 C —2

 

iCa 4.1 4.2 4.5 0.08 P < 0.01 P < 0.05
K 18.0 18.5 19.0 0.49 P = 0.57 P = 0.49
Na 337 338 338 1.3 P = 0.64 P = 0.81
Cl 380 384 383 2.5 P = 0.22 P = 0.68

 

C -1:+14 vs. 0 and -15 DCAD, C - 2:0 vs. -15 DCAD

Plasma Ionized Ca (mg/d1)

24

 

 

 

 

 

 

 

 

- l —O- Primiparous Control l- _ * 1

1-0- Primiparous ODCAD 1-_-1

. --e- Primiparous -15 DCAD, 1

3.5 1 ,,,,,,,,,,,,,,,,,,,, 1 _______ , _ m . _ , _______ ! +Multiparous Control l_ _ _ . 1
l + Multiparous 0 DCAD ! 1

3-4’1'”"“”* """""""" "‘1—e—Multiparous-15DCAD1‘ j
l

3.2 . eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee — - — «

3 0 ——— 4— —+———+ B~ i ref—e — + ————.——— , ———4——— t -. —
-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16

Days relative to parturition

Figure 1. Parity by treatment effect on peripartum plasma iCa concentrations (mg/d1)
(P < 0.05) SEM = 0.11.
Plasma hydroxyproline, PTH, and calcitriol

Plasma samples were analyzed at -14, -7, -2, -1 d pre-calving, and 0, 12 h, 1, 2, 3
d post-calving for concentrations of hydroxyproline. Hydroxyproline was affected by
day, parity, and parity by day (P < 0.001, Figure 2), but not by treatment (Table 5).
Hydroxyproline is an indicator of bone resorption (Leclerc and Block, 1989; Wang et al.,
1994). The lack of a treatment effect on hydroxyproline was unexpected, and suggests a
lack of increased bone mobilization with the inclusion of anionic salts. If so, the

increased dietary Ca was the source of increased plasma iCa seen with the 0 DCAD and -

25
15 DCAD diets. Whether the acidifying effects of anionic salts helped potentiate the

effects of dietary Ca on plasma iCa cannot be determined in this study because dietary Ca
varied among treatments as well as DCAD. Oetzel et al. (1988) showed in a 2 X 2
factorial experiment that dietary Ca intake (53 g/cow per (1 vs. 105 g/cow per d) alone did
not affect serum concentration of iCa, but that higher dietary Ca concentration with a
negative DCAD did lower the incidence of hypocalcemia. Leclerc and Block (1989)
showed an increase in plasma hydroxyproline with reduced DCAD when using a constant
dietary Ca concentration of 1.3 %. In the present study, the potential positive effect of a
reduced DCAD on bone resorption may have been masked because of the confounding of
DCAD with different dietary Ca concentration (Control = 0.44 %, 0 DCAD = 0.97 %,
and -15 DCAD = 1.52 %). The results of Oetzel et al. (1988) indicate that increased
plasma iCa concentrations in cows fed the anionic salts was due to both the DCAD and
the dietary Ca concentration.

Hydroxyproline concentrations were higher for primiparous cows than
multiparous cows, suggesting greater mobilization of bone (P < 0.001 SEM = 3.45)
(Figure 2). Older cows may not be able to respond as well as primiparous cows to Ca

challenges around the time of calving because of a decreased ability to mobilize bone.

26

 

 

 

 

 

 

 

4
E
g. +primiparou’J
g +muttiparous
-o ____4 L,
5‘
0. Q5 ______________________________________

0 :

-15 -1O -5 O 5
Days relative to calving

Figure 2. Parity by day effects on plasma hydroxyproline concentrations (ug/ml)
(P < 0.001) SEM = 3.45.

Table 5. Treatment effects on plasma hydroxyproline' (ug/ml), PTH2 (pg/ml), and
calcitriol3 (pg/m1) concentrations.

 

 

Item Control 0 DCAD -15 DCAD SEM C — 1 C - 2
Hydroxyproline 2.37 2.39 2.30 0.15 P = 0.87 P = 0.61
PTH 46.7 52.0 23.5

log PTH 3.30 3.12 2.70 0.16 P < 0.05 P = 0.06
Calcitriol 78 57 41

log Calcitriol 4.13 3.74 3.55 0.09 P < 0.001 P = 0.12

 

C - 1: +14 vs. 0 and -15 DCAD, C - 2:0 vs. -15 DCAD
' Means of samples taken at 14, 7, 2, 1 d prepartum and 0, 12 h, 1, 2, 3 d postpartum.
2 Means of samples taken at 2, 1 d prepartum and 0 h, 1, 2 d postpartum.
3 Means of samples taken at 7, 1 d prepartum and 0 h, 1 d postpartum.

Plasma samples were analyzed at -2, -l d pre-calving, and 0 h, 1, 2 d post-calving
for concentrations of PTH. Plasma PTH concentrations were transformed to log base 10
for analysis because of heterogeneous variances. PTH concentrations were affected by

treatment (P < 0.05), treatment by day (P < 0.05), day and parity by day (P < 0.001)

(Table 5, Figure 3 and 4). Cows fed Control had higher PTH concentrations than the

27
average of cows fed the 0 and -15 DCAD diets (P < 0.05), and there was a trend for

higher PTH concentrations with the 0 DCAD diet than in the -15 DCAD diet (P < 0.1).
Decreased plasma PTH concentrations with 0 and ~15 DCAD diets around the time of
calving (-2 to +2 (I) was consistent with the plasma iCa concentrations, the reason for the
elevated PTH with 0 and —1 5 DCAD compared with Control cannot be explained. The
higher PTH concentration of cows on the 0 DCAD (Table 5 and Figure 3) is not
consistent with the relative ranking of iCa concentrations among treatments and may be
due to high variability or bias (heterogeneity of variances) in PTH data. D’Amour et al.
(1986) showed that in calves, dogs and man, plasma PTH concentrations were in close
inverse relation with plasma iCa concentrations. This close relationship between plasma
concentrations of PTH and iCa is supported by the results of others (Jonsson et al., 1980;
Kumar 1980; Ramberg et al., 1967).

The decreased plasma PTH of cows fed the 0 and -15 DCAD diets is one more

indicator of the better Ca status of these cows compared with the Control cows.

28

 

+Controlmfi
+0 DCAD
+45 DCAD“.

 

 

 

Plasma PTH (ngrnl)

 

 

 

Daya relative to calving

Figure 3. Treatment by day interaction on plasma PTH concentrations (pg/ml).

 

140

120 .

100<

 

+Primiparous 1
+Multiparous 1

 

 

Plasma PTH (pglmi)

 

 

 

 

-2 -1 o 1 2
Days relative to calving

Figure 4. Parity by day interaction on plasma PTH concentrations (pg/ml).

Plasma samples were analyzed at -7, -1 d pre-calving, and 0 h, 1 d post-calving

for concentrations of calcitriol. Mean plasma calcitriol concentrations for these sample

29

points were transformed to log base 10 for analysis because of heterogeneous variances.
Calcitriol was affected by treatment, parity, and day (P < 0.001). The interactions of
parity by treatment, treatment by day, and parity by day were also significant (P < 0.05)
(Figures 5 and 6). Table 5 gives the actual calcitriol concentrations for the Control and
treatment diets. Cows fed the Control diet had higher calcitriol plasma concentrations
than cows fed the 0 and -15 DCAD diets (P < 0.001), but plasma calcitriol concentrations
of cows fed 0 DCAD did not differ from the -15 DCAD (P > 0.1). The lower plasma
calcitriol concentrations with increased plasma iCa concentration is consistent with the
results of others (Bushinsky et al., 1982; Goff, 1992a; Jonsson, 1978; Kumar, 1980).
Gaynor et al. (1989) however, reported that anion diets increase plasma calcitriol before
calving. However, the increase in calcitriol that they saw was most likely due to an
increase in Ca flux and the fact that the cows fed the anion diet actually had lower plasma
Ca concentration prior to calving than their cation group (the same time period that
calcitriol was analyzed for). The higher plasma PTH and calcitriol concentrations of
Control cows (which were also hypocalcerrric) is also consistent with the thought that
bone and renal tissues of hypocalcerrric cows may have reduced sensitivity to the effects
of PTH and calcitriol (Block, 1992; Goff, 1992a). The higher calcitriol concentrations of
Control cows are also consistent with the positive effects of plasma PTH concentration on

calcitriol concentration (Block, 1992; Jonsson, 1978).

160

 

‘w-4

1201

Plasma Calcitriol (pg/ml).
8
-+

____________________

 

 

 

 

 

 

 

Days relative to calving

Figure 5. Treatment by day effects on plasma calcitriol concentrations (pg/ml) of
primiparous cows in the peripartum period.

   
 

d
£1001. -7. 7
: Miamirar‘“
goo.-- - ._ ....... ______ ____ 11+ODCADI
3 ~L:*:5°°A°
3.. __________ _ 1

l

1

4o _______________________________________________________ 1

 

 

0* ‘ ~—1—— 7 + — —+————— +————————+—- + A
7 -6 5 .4 3 -2 1 o 1
Days relative to calving

Figure 6. Treatment by day effects on plasma calcitriol concentrations (pg/ml) of
multiparous cows in the peripartum period.
Acid-base balance: urine pH, plasma pH, plasma pCO” and plasma HCO3

Cows fed the Control diet had higher urine pH than cows fed the 0 and -15 DCAD
diets during the prepartum period (P < 0.005) (Table 6). Cows fed the 0 DCAD diet had
higher urine pH than cows fed the -15 DCAD diet (P < 0.005). The decreased urine pH
indicates that the acid-base status of the cows was altered by the addition of anionic salts
to their diets (Goff, 1992a; Tucker et al., 1988).

Plasma pH and pCO2 were not affected by treatment in the prepartum period (P =
0.18 and P = 0.15 respectively) (Table 6), although numericallythey were both lower for

cows fed the -15 DCAD diet. Plasma HCO3 was lower in cows fed the -15 DCAD diet

32
than cows fed the 0 DCAD diet (P < 0.001), and Control cows had higher plasma HCO3

concentrations than did 0 DCAD and —1 5 DCAD (P < 0.001) (Table 6).

According to the Henderson Hasselbach equation; blood pH = 6.1 +
loglo[HCO3/(.03 X pCOz)]. Changing either plasma HCO3 or pCOz, will thus have an
immediate affect on plasma pH if there is not a concurrent change in the other. In this
study as in others (Tucker et al., 1991; Tucker et al., 1988), plasma HCO3 was decreased
with the inclusion of anionic salts but pCO2 was not. In the present study, HCO3 was not
reduced until urine pH was acidic and DCAD was negative. The necessity for urine pH
to be acidic and DCAD to be negative before plasma HCO3 is affected is not supported by

Tucker et al. (1991).

Table 6. Effects of treatment on acid-base status of cows up to 1d prepartum.

 

 

Item Control 0 DCAD -15 DCAD SEM C — 1 C — 2
Urine pH 7.98 7.30 6.21 0.082 P < 0.001 P < 0.001
Plasma pH 7.58 7.58 7.56 0.011 P = 0.29 P = 0.12
Plasma pCOz, mm Hg 25.8 25.3 24.0 0.67 P = 0.16 P = 0.16
Plasma HCO3, mM 24.1 23.5 21.1 0.38 P < 0.001 P < 0.001

 

C - 1: +14 vs. 0 and -15 DCAD, C - 2:0 vs. -15 DCAD

At the time immediately after calving (0 h, 12 h, 1 d), plasma pH was affected by
treatment (P<.05) with Control and 0 DCAD having higher pH than -15 DCAD (Table 7).
The difference in pH soon after calving may have been due to a lag effect of higher
anions being present in —1 5 DCAD cows as compared to Control and 0 DCAD cows.

Plasma pCO2 and HCO3 were not affected by treatments (Table 7).

33

Table 7. Effects of treatment on acid-base status of cows immediately after calving (0h,
12b, and 1d).

 

 

Item Control 0 DCAD -15 DCAD SEM C — 1 C — 2

Plasma pH 7.6 7.6 7.5 .01 P < 0.05 P < 0.05
Plasma pCOz, mm Hg 26.1 26.0 27.5 .98 P = 0.59 P = 0.28
Plasma HCO3, mM 25.3 24.4 23.5 .60 P = 0.08 P = 0.30

 

C - 1: +14 vs. 0 and -15 DCAD, C - 2:0 vs. -15 DCAD

Dry matter intake

Prepartum DMI data were analyzed as sets for 3 d before to 2 d after cows were
offered the experimental diets, as repeat measures during the 3 wk prepartum period, and
as average DMI by each cow across the 3 wk prepartum period.

Analyzing the data for -3 to +2 (I on treatment diet, showed a significant effect of
parity (P < 0.001), day (P < 0.001), and treatment by day (P < 0.001) on DMI (Table 8,
Figure 7, respectively). There was also a trend for a parity by day (P = 0.07) and
treatment (P = 0.07) effect on prepartum DMI. DMI was not affected (Figure 7). This is
likely due to the 3 d before dietary treatments were administered being included in the
analysis. A treatment by day interaction existed for DMI, showing that —15 DCAD

decreased DMI over time, whereas DMI was not affected by 0 DCAD or Control

(Figure 7).

Table 8. Effect of parity on DMI (kg/d) from -3d to +2d on treatments.

 

Item Primiparous Multiparous SEM P

 

DMI 10.8 16.7 0.55 0.001

 

34

 

 

rI-4— Control-‘1 “1
+0 DCAD 1
1:11:92?

Dry latter Intake (kg)

 

 

 

Daya relative to treatment initiation

Figure 7. Effect of treatment by day interaction on DMI (P < 0.001) SEM = 0.69.

When analyzing the DMI data for 3 wk prepartum to calving, week (P < 0.001),
parity by week (P < 0.001), parity (P < 0.001), and treatment (P < 0.005) were all
significant (Table 9, Figure 8). Over the 3 wk prepartum period, primiparous cows had
mean DMI of 9.6 kg/d compared with 14.1 kg/d DMI for multiparous cows

(SEM = 0.56).

Table 9. Treatment effects on prepartuml DMI (kg/d), BW (kg), and BCS.

 

 

Item Control 0 DCAD -15 DCAD SEM C - 1 C — 2
Prepartum
DMI 12.9 12.5 10.2 0.55 P < 0.05 P < 0.01
BW 733 729 701 5.2 P < 0.01 P < 0.001
BCS 3.88 3.77 3.63 0.084 P = 0.11 P = 0.20

 

C - 1: +14 vs. 0 and -15 DCAD, C - 2:0 vs. -15 DCAD
' Prepartum period equals -3 wk to calving for DMI. Prepartum BW and BCS are
at -1 wk adjusted for pretreatment covariate.

35

 

1i Pnfmigarofi
lIMultiparous;

Dry Mathr Intake (kgld)

 

 

 

-3 -2 -1
Weeks Prepartum

Figure 8. Parity by week interaction on DMI (P < 0.001) SEM = 0.56.

Cows fed Control diet had higher DMI than did cows fed the 0 and -15 DCAD
diets (P < 0.05) during the 3 wk prepartum period (Table 9). Cows fed the 0 DCAD diet
had higher DMI than did cows fed the -15 DCAD diet (P < 0.01). This DMI depression
in the - 15 DCAD diet is consistent with the experimental hypothesis. These results are
also consistent with the literature in that anionic salts are not palatable and dietary
formulation must account for this reduction in DMI (Beede, 1995; Goff, 1992a). The
reduced DMI seen in the prepartum period might be expected to affect body weight and
BCS.

Postpartum DMI was analyzed using the same model as prepartum DMI with 10
wk of data per cow. Treatments had no effect on postpartum DMI (P > 0.42). There was
a trend for a treatment by parity interaction (P = 0.08) with the 0 DCAD tending to have

greater DMI than the -15 DCAD diet in multiparous cows.

36

Body condition score and body weight

BCS and BW (kg) at -1 wk prepartum were analyzed using the respective
pretreatment mean values as covariates (Table 9). Prepartum BCS was not affected by
treatment. Prepartum BW was higher for Control cows than for cows fed anionic salts
and higher for cows fed 0 DCAD than -15 DCAD (733, 729, and 701 respectively,
P < 0.001, SEM = 5.23). Thus although all cows were gaining weight prior to calving,
DMI depression in the -15 DCAD diet did lower BW at -1 wk prepartum compared with
the 0 DCAD diet.

Postpartum, BCS was only affected by parity and week. Body weight was
affected by week, parity by week, and parity (P < 0.005). There was a trend for a

treatment by parity interaction (P = 0.1).

Energy balance

Prepartum (-2 and -1 wk) energy balance was lower for cows fed the -15 DCAD
diet than for cows fed the 0 DCAD diet (EB = 0.8 and 5.5 Meal/day, P < 0.01, SEM =
1.14) (Table 10). Reduced energy balance prepartum, resulted from the depression in
BW and DMI seen in the prepartum period with cows fed the -15 DCAD diet compared
with cows fed the 0 DCAD diet, but also indicated all treatments maintained a positive
energy balance in the prepartum period.

Postpartum (2 - 9 wk) energy balance was not affected by treatment (Table 10).

37
Table 10. Energy balance (Meal/d) as affected by treatment, prepartum and postpartum.

 

 

Item Control 0 DCAD -15 DCAD SEM C — 1 C — 2
Prepartum 5.1 5.5 0.8 1.14 P = 0.17 P < 0.01
energy balance

Postpartum -2.1 -.74 -1.2 1.2 P = 0.45 P = 0.80

energy balance

 

C - 1: +14 vs. 0 and -15 DCAD, C - 2:0 vs. --15 DCAD

NEFA concentrations in plasma
Treatment had no effect on NEF A concentrations (P > 0.9) (Table 11). NEF A
concentrations were affected by day, pre and postpartum (Figure 9). NEFA
concentrations increased as parturition approached, with peak NEFA concentrations
occuring 1 d postpartum. NEF A concentrations gradually declined afier parturition.
Diets were formulated to meet the energy and nutrient requirements of a 612 kg
cow consuming 10 kg of DMI. These results emphasize the need for the formulation of a

nutrient dense diet for the prepartum cow when feeding anionic salts.

Table 11. Plasma NEFA (uM) and IGF-I (ng/ml) concentrations by treatment.

 

 

Item Control 0 DCAD -15 DCAD SEM P
NEFA 550 510 530 53.7 0.9
Prepartum

IGF-I 216 195 193 13.4 0.441
Postpartum

IGF-I 128 126 112 16.3 0.738

 

 

1200 .7. .#. _. __ __ L7 1

 

1 {Primiparous .
+Multiparous 1

 

 

NEFA In plaarna (uM)

 

 

-21 -16 -11 -6 -i 4 9 14 19
Daya relative to calving

Figure 9. Plasma NEF A concentrations (uM) before and after parturition (P < 0.001)
SEM = 74.2.

Plasma IGF-I

IGF-I concentrations for prepartum and postpartum samples were analyzed
separately. Treatment had no effect on plasma IGF-I concentrations prepartum or
postpartum (P > 0.4) (Table 11). Prepartum IGF-I concentrations were affected by day
and parity (P < .005), with IGF-I concentrations decreasing as parturition approached and
IGF-I concentrations being lower in multiparous cows than in primiparous cows (165 vs.
237 uM respectively, P < 0.005, SEM = 15). Postpartum IGF-I concentrations were only
affected by parity (P < 0.005), with multiparous cows having lower plasma
concentrations than prinriparous cows (89 vs. 155 uM respectively, P < 0.005, SEM =
13).

Plasma IGF-I concentrations have been positively correlated with energy balance

in heifers and cows (Sharma et al., 1994; VandeHaar et al., 1995; Zurek et al., 1995).

39

Plasma IGF-I concentrations decreased as parturition approached, which is consistent

with the decrease in DMI as calving approached.

Calf weight

Calf weight was higher for multiparous than primiparous cows (P < 0.05). There
was a trend for cows fed the 0 DCAD diet to have larger calves than cows fed the ~15
DCAD diet (P = 0.08) (Table 12).

As mentioned previously, DMI and body weight gain were both suppressed by the
~15 DCAD diet. Whether this contributed to the reduced calf weights in the ~15 DCAD

diet cannot be determined.

Table 12. Treatment effects on calf weight (kg).

 

Item Control 0 DCAD ~15 DCAD SEM C — 1 C — 2

 

Calf Weight 42 46 42 1.32 P = 0.27 P = 0.08

 

C ~ 1: +14 vs. 0 and ~15 DCAD, C ~ 2:0 vs. ~15 DCAD

Milk yield and components

There were no effects of treatments on milk yield or any milk components when
analyzed for 2 through 9 wk postpartum (Table 13). There was a treatment by week
effect for 4% FCM yield (P < 0.05, Figure 10) with cows fed the ~15 DCAD diet
appearing to start out at a lower level of production. Figure 10 shows that the mean F CM
for the Control group dropped sharply after wk 6 postpartum. One cow in the Control

group dropped significantly in production during this time period due to mastitis.

4O
Removal of this cow did not change the statistical outcome or make any meaningful

change to the graphs of the results, therefore that cow is included in the data analysis.

Table 13. Treatment effects on daily milk yield and components.

 

 

 

Item Control 0 DCAD ~15 DCAD SEM C — 1 C — 2
Milk (kg) 37 37 34 1.3 P = 0.45 P = 0.15
FCM (kg) 36 36 34 1.5 P = 0.70 P = 0.23
Fat % 3.8 3.8 3.8 0.12 P = 0.75 P = 0.98
Fat (kg) 1.4 1.4 1.3 0.07 P = 0.87 P = 0.34
Protein % 2.8 2.9 2.9 0.05 P = 0.56 P = 0.80
Protein (kg) 1.0 1.1 1.0 0.04 P = 0.64 P = 0.14
Lactose % 4.2 4.3 4.3 0.07 P = 0.09 P = 0.65
Lactose (kg) 1.5 1.6 1.5 0.06 P = 0.92 P = 0.13
SCC 392 308 444 114 P = 0.91 P = 0.41
C ~ 1: +14 vs. 0 and ~15 DCAD, C - 2:0 vs. ~15 DCAD
8 W601“?
g +0 DCAD
3 +-15 DCAD
IL

 

 

 

—.Pfi— u+ — —+——— -- -- +——~— +-———+—~ ——4
3 5 6 7 8 9
Weeks Postpartum

Figure 10. Treatment by week interaction on 4% FCM (kg), (P < 0.05) SEM = 0.938.

41
Any prepartum dietary effects on milk yield and components would most likely

occur shortly after parturition. Milk yield and component data were thus also analyzed
for treatment effects from 2 to 5wk postpartum. Milk yield tended to be lower for cows
fed the ~15 DCAD diet than for cows fed the 0 DCAD diet (milk yield = 32.6 and 36.6
kg/d respectively, P = 0.07, SEM = 1.47). F CM yield did not differ (P = 0.13).

Milk yield and FCM yield responses were not consistent with the literature
(Beede, 1995; Block, 1984). Block showed that cows fed an anionic diet prepartum had
higher (7% increase) 305-d milk yields than cows fed an cationic diet. Verdaris and
Evans (1974) showed that when looking at Ca concentrations in the diet, cows with high
Ca (2.48%) during an 86~d dry period and 84~d milk period produced the most milk
compared with cows with low-low (prepartum-postpartum, 0.23%), low-high, or high-
low dietary Ca concentrations. Increasing inclusion of anionic salts in the present
experiment caused a lower DCAD as well as greater dietary Ca concentration. These
studies suggest that both effects could increase milk production for the ~15 DCAD
treatment group. However, in the present experiment cows on —1 5 DCAD prepartum
appeared to have lower FCM production at wk 2 postpartum (Figure 10). Lower milk
yield at 2 wk postpartum may have been due to the suppressed DMI during the late dry

period with the increased anionic salts (~15 DCAD).

Incidence of Metabolic Disorders
Table 14 shows the incidences of the various metabolic disorders that were
recorded during the experiment. Analysis with Fisher’s Exact Test (2-Tail) showed that

there were no differences in the incidences of any of the metabolic disorders as affected

42
by treatment. Cows having twins were not included in this analysis. There was a trend

(P = 0.09) for a difference in milk fever incidence, with all incidences of milk fever
occuning in cows (three of nine multiparous cows) fed the 0 DCAD diet. Number of
cows per treatment group was too small to reliably detect differences in metabolic

disorder.

Table 14. Incidence of metabolic disorders.

 

 

Item Parity Control 0 DCAD ~15 DCAD
Milk Fever Primiparous 0 0 0
Multiparous 0 3 0
Ketosis Primiparous 5 5 4
Multiparous 2 0 2
Displaced Abomasum Primiparous 2 1 3
Multiparous 0 0 1
Retained Placenta Primiparous 2 1 0
Multiparous 3 1 1
Metritis Primiparous 2 2 0
Multiparous 2 0 1

 

SUMMARY AND CONCLUSIONS

Milk fever (hypocalcemia) has been defined as plasma iCa concentrations 5 4
.mg/dl (Beede et al., 1992; Oetzel et al., 1988). Low plasma iCa concentrations cost the
US. dairy industry over $120 million/year in the cost of health disorder treatments and
secondary problems (Goff and Horst, 1990). The feeding of anionic salts to the
prepartum cow is becoming a popular method to control hypocalcemia. The focus of this
experiment was to determine if a lower inclusion of salts (0 DCAD) would have the same
positive effects on Ca status in the dairy cow as that currently recommended (~10 to ~15
meq/ 100 g DM DCAD), while improving DMI. In order to accomplish this change in
DCAD, an anionic salts pack was developed for the ~15 DCAD diet and included at a
lower rate for the 0 DCAD diet. This method of adjusting the DCAD would be most
consistent with how a dairy producer would adjust the DCAD on the farm. The one
confounding factor with this type of approach was that the dietary Ca concentrations
increased as more anionic salt pack was fed.

The results of this study are not completely consistent with the hypothesis or
alternative hypothesis. Prepartum DMI was depressed in the ~15 DCAD treatment
compared with 0 DCAD treatment. Cows fed the ~15 DCAD diet had lower BW 1 wk

prepartum compared with cows on the 0 DCAD diet. Prepartum energy status was

43

 

44
decreased for the cows fed the ~15 DCAD diet, but was still positive. Plasma NEFA,

IGF—I, and hydroxyproline concentrations were not affected by treatment. Decreasing
DCAD increased plasma iCa concentrations both prepartum and in samples shortly after
calving (0 h, 12 h, 1 d). There was a parity by treatment effect on plasma iCa
concentrations with primiparous cows being unaffected by treatment, whereas iCa increased
as DCAD decreased in multiparous cows. Acid-base status of the cows was affected by
treatment, with cows fed the ~15 DCAD diet having lower plasma HCO3 concentrations
than cows fed the 0 DCAD diet. Urine pH also decreased with decreasing DCAD during
the treatment period. Control cows had higher PTH and Calcitriol concentrations than
cows on the 0 and ~15 meq/ 100 g DM. There was a treatment by week effect on F CM yield
(2 through 9 wk) with cows fed ~15 DCAD starting at a lower level of milk production.

The results of this experiment indicate that anionic salts should be fed to prepartum
cows at a level to prevent hypocalcemia and maintain DMI. This will require careful diet
formulation and careful monitoring of urine pH by dairy producers to evaluate the
effectiveness of anionic salts. The lack of hypocalcemia, along with the negative effects
of anionic salts on DMI, suggests that dairy producers should not feed anionic salts to
primiparous cows.

The present study indicated that urine pH can be used as an indicator of the acid-
base status of the cow. The urine pH values found in this experiment will give dairy
producers a good tool for estimating the success they are having in reducing the DCAD
for their close-up cows. One strategy for producers, would be to formulate diets at 0

DCAD and then titrate urine pH to 7.0 by including more or less anionic salts in the diet.

 

45

This may insure a mild metabolic acidosis that would result in less DMI depression than
the ~15 DCAD diet did in this experiment.

Future research will need to look into separating out the effects of DCAD and
dietary calcium concentration on plasma iCa concentrations. Research will also need to
look at ways to better determine how anionic salts increase plasma iCa levels. Specifically
how much of the increased plasma iCa is from intestinal absorption or bone resorption, and

does one source decrease parturient hypocalcemia to a greater extent.

 

APPENDIX

APPENDIX
Procedug for IGF-I analysis

Iodination was carried out using 2 ug recombinant pure human IGF-I (Bachem Inc.,
Torrance, CA) dissolved in 10 ul 0.1 N HCl . Thirty-five ul 0.5M phosphate buffer
solution (PBS) was added and mixed. One mCi 125I radionucleotide in 0.1 M NaOH
(DuPont , Boston, MA) was added and mixed. Chloramine T (25 ul; 1 ng/ml) was added
using a Hamilton syringe and mixed. Fifteen seconds later, 50 ul sodium metabisulfite (5
mg/ml) were added and mixed. Twenty seconds later, 100 ul of transfer solution were
added and mixed. This reaction mix (about 100 to 200 ul total volume) was then carefully
transferred to a column bed (see Appendix for description). Twenty-five 0.5-ml fractions
were then collected from the column using elution buffer. The column was used to separate
bound (i.e., incorporated) 125I from free ”51. Ten ul from each fraction were counted for 0.1
min. Fractions comprising the first peak (bound 12’1) were saved for use in the IGF-l assay.
Free 125I was discarded. Fractions containing the first peak were tested for TCA precipitable
counts to determine if the ”’1 had indeed labeled IGF-I. A test for total binding and specific
binding was run on the fractions to determine the level of specific binding in each fraction.

Serum samples for IGF-l assay were extracted by an ethanol acid procedure (Bruce
et al., 1991) with modifications by Shanna et al. (1994) to remove IGF-I binding proteins.
To each 50 ul of serum sample, 12.5 ul of formic acid and 250 ul absolute ethanol were
added. Samples were mixed, by vortex, and allowed to stand at room temperature for 30
min. Samples were then centrifuged for 30 min at 4°C at 600 x g. An aliquot of
supernatant (50 ul) was removed and diluted to 1.05 ml total volume using neutralizing
buffer. The total dilution was 1:131.25. Of this extract, 50 111 in triplicate were used for the
IGF-I assay.

Standard curve consisted of 0, 10, 20, 30, 40, 60, 80, 100, 150, 200, 300 and 400
pg/tube of diluted standard A. Fifty ul of serum extract were pipetted in triplicate assay
tubes. Tubes labeled NSB contained no first antibody. Tubes labeled TC were kept empty.
The final volume of all assay and standard tubes were brought to 200 111 with assay buffer.

To these assay tubes, 250 ul of first antibody (diluted 1:40,000) was added (except
to TC and NSB samples), tubes were mixed, by vortex, and all samples were incubated at
4°C overnight. To NSB tubes, 250 ul of assay buffer was added. On (I 2, 18,000 to 20,000
cpm iodinated IGF-I was added to each tube including TC tubes. Tubes were stored at 4°C
for another 48 h. On (1 4, 200 ul/tube of Staph A (dissolved in 39 ml assay buffer) were
added to all tubes except TC tubes. Samples were mixed, by vortex, and stored at room
temperature for 4 b. Two ml of assay buffer were added to each tube and all tubes were
centrifuged for 30 min at 4°C and 3000 rpm. The supernatant was discarded properly. All
tubes were dried with pellet intact and then counted for 1 min.

Plasma IGF-I content was calculated using the standard curve developed from the
standards.

46

47

Buffers and reagents for RIA of IGF-I

Recombinant human IGF-I:
rhIGF-l from (Bachem, Inc., product #DGR012), 50 ug is dissolved in 1 ml of 10
mM acetic acid solution and made aliquots of 40 ul = 2 ug into labeled Eppendorf
tubes, dried in Savant and stored at ~20C until used.

International reference standard of IGF-I from World Health Organization (WHO):
Obtained 3.1 ug IGF-I containing ampoule from National Inst. of Biological
Standards and Control (NIBSC). Dissolved the total content in 1 ml of 0.05M HCl
and transferred to already tared 50 ml orange top tube and make final weight of
solution to 15.5 g using 0.03 M phosphate buffer pH 7.5. Make 100 111 aliquot and
store at ~20 C.

8% Bovine Serum Albumin (BSA):
0.8 g BSA Qs to 10 ml total volume with water, pH at 7.5

Protein A positive Staph. aureus cells:
Dissolve 1g of Staph A (BM 100 061) into 10 ml distilled water. And make into 1
ml aliquot. Store at ~20 C.

Assay buffer:
It is 0.03 M sodium phosphate, .01 M EDTA, 0.02% protarrrine sulfate, 0.02%
sodium azide and 0.05% Tween-20 with final pH 7.5.

Elution buffer:
0.03 M phosphate bufler with 0.25% BSA.

Neutralizing buffer pH 7.5:
NaQI-IPO4 0.11 moles/liter; NaCl 0.154 moles/liter; NazEDTA 0.01 mol / liter;
Sodium azide 0.015 moles/liter; Solution Tween-20 .5%w/v. [weigh 15.62 g
Na2HP04, 9.0g NaCl, 3.7 g EDTA, 0.975g Na-azide, and add 0.5 ml of 10%
solution of Tween-20.

Transfer solution:
Weigh 100 milligram of potassium iodide (KI), and weigh 1.6 g sucrose. Dissolve
them in double distilled water to make total volume=10 ml. Make 1 ml aliquot and
store at ~20C.

PBS solution:
It is .5 M sodium phosphate buffer pH 7 .5.

48

Standard curve for IGF-I assay:

20 nanograms of rhIGF-I (in 100 ul PBS) was made to a total volume of 200 ul with
assay buffer. 150 ul of this solution (15 nanograms) was removed and made to a
total volume of 3 ml with assay buffer. This solution was called A (5 picograms/ul).
Out of the 3 nrl of solution A, 1 ml was removed and taken to total volume of 5 ml
with assay buffer. This second solution was called B (1 picogram/ul).

mparafion of Column:

1)

2)
3)

4)

Prepare a 10 ml column using 2 parts ofcoarse and 1 part ofmedium sized
Sephadex G-50 swollen in .03 M PBS. Use yellow pipet tip and glass wool
to regulate flow. Run plenty of degassed elution buffer through the column.
Regulate the flow at about 1 ml/5 min.

Run 1 ml of 8% BSA to coat the column material with protein.
Prepare a fresh solution of sodium metabisulfite 5 mg/ml.
Prepare fi'esh solution of Chloramine T, 1 mg/ml. Make in dark, wrapped

with foil. Weigh 18 mg and dissolve in 18 ml .03 M sodium phosphate
buffer with no BSA.

Determining TCA Precipitable Count:

1)

2)
3)
4)
5)
6)

7)

50 ul fiom each fraction comprising the peaks was placed into a series of
duplicate 12x75 tubes, keeping track of cpm that was added to tubes from
each fraction.

800 ul of ice cold 8% BSA was added.

200 ul of ice cold 100% TCA was added.

All samples were chilled on ice for 30 minutes.

1~2 ml of assay buffer was added.

Samples were spun at 300 rpm for 30 minutes.

Supernatant was transferred to a new set of tubes using a 1 ml pipet

49

8) Both sets of tubes were corked and counted in the gamma counter.

9) Total cpm, cpm in supernatant, and % cpm in pellet were calculated.

10) The free iodine peak was determined.

11) Fractions comprising free iodine peaks were discarded as FREE I~waste.

Test for Total Binding and Sflific Binding

For each fraction of bound IGF-I, an assay was set up to test the actual specific binding.
The table below shows an example of the specific binding assay.

Fraction No.

TC (about 15000cpm)
NSB (N o 1‘it AB)
Zero, 200 111 buffer

 

 

 

 

 

50 ul=50 pg IGF-I in
200 ul elution buffer

 

100 ul=100 pg IGF-I
in 200 ul elution
buffer

 

 

 

 

 

 

 

 

Note: Tubes were labeled as:
NSB got no first AD but everything else.
TC got nothing but iodinated IGF-I from its respective fraction.
Zero got 200 111 buffer and then everything else.

 

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