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Wan-m

 

EFFECT OF PREPARTUM DIETARY ENERGY DENSITY AND
PROTEIN CONTENT ON PERIPARTUM BODY FAT MOBILIZATION.
MILK YIELD, HEALTH PERFORMANCE AND PROTEIN TURNOVER IN

HOLSTEIN cows
By

Galal Moustafa Yousif

A THESIS

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

MASTER OF SCIENCE

Department of Animal Science

1 995

ABSTRACT

EFFECT OF PREPARTUM DIETARY ENERGY DENSITY
AND PROTEIN CONTENT ON PERIPARTUM BODY FAT MOBILIZATION.
MILK YIELD, HEALTH PERFORMANCE AND PROTEIN TURNOVER IN
HOLSTEIN COWS

Bv

Galal Moustafa Yousif

Eighty Holstein cows ln=80; 4O primiparous and 40 multiparousl
were used, in a complete randomized block design (20 blocks). where cows
were blocked randomly by parity and expected calving date. Dry cows four
weeks from their expected calving dates were fed one of the four
experimental diets: 1) Low energy and low protein lLLl..2l Medium energy
and medium protein (MM). 3) Medium energy and high protein (MH). 4) High
energy and high protein (HHI. After calving, all cows were fed the same
diet according to NBC recommendations. Cows that were fed the HH diet
had lower blood NEFA concentrations prepartum compared to the cows that
were fed the LL diet IP<O.1I. Treatment diets did not affect feed intake,
.body weight, body condition score, milk yield. Liver TG concentrations

were affected by treatment diets. The results of this study suggest that

increased energy and protein for dry cows decreases plasma NEFA

concentrations two weeks prepartum.

To my sweet little sister Assal Yousif.‘
and to my mother Awatif Sabra, her belief in family and in education

provided me with the foundation necessary to engage in educational

inquiry.

iv

W

I wish to extend my sincere gratitude to Dr. Roy S. Emery, my
graduate committee chairman and my major advisor for his encouragement.
guidance and support. His vast knowledge of dairy cattle nutrition has
provided the necessary expertise for completion of this study.

I also wish to express my appreciation to the members of my
graduate committee: Dr. Micheal Vandehaar, whose counsel served me to
understand my goals, and Dr. Andy Skimore. Special thanks are due” to Dr.
John Kaneene for his continuous encouragement and valuable advice.

I especially want to thank Dr. A. Y. M. Nour and Dr. Robert Cook for
their valuable academic and moral support. My words cannot express
enough appreciation to Dr. Balkrisha Sharma and Jim Liesman for their
. academic advice and help throughout the course of my graduate program.

I further wish to express my sinceregratitude to Dr. Werner Bergen,
Ms. Gwen El-Khalifa. A. Yacoub, David Main, Dave O'Daniel, Dewey
LonguSki. Bob Kreft, Bob Story, the dairy farm crew. and my fellow
graduate students Mee-chainn ’Yung and Paul Dyk. To my family and the
Sudanese community in Lansing lam indebted for their. help in different

ways that made it possible for me to complete this degree.

V

Special thanks are due to Miss. Yasmeen Abusamra, my fiance, for

her long distance encouragement and support for her trust in me.

vi

TABLE OF CONTENTS

LIST OF TABLES ................................................................... ix
LIST OF FIGURES......................' ...................... . ..................... x
INTRODUCTION ............. 1
LITERATURE REVIEW .................................. , ...... _ .............. 3
Nutrient Requirements ............ . ....................................... 6
Carbohydrate ................................................................. 6
Protein ......................................................................... 7
NRC Recommendations .................................................. 8
Feed Intake"; .............................................................. 10
Ketosis. Fatty Liver. and Fat Cow Syndrome ........... ' ........ 1 3
Fatty Liver .......... , ................. 16
Fat Cow Syndrome ...................................... . ................ 20
Metabolism of Glucose and lipids in the Liver
of Dairy Cattle ............................................................ 22
Gluconeogenesls .................................................. 22
Precursors and Pathways..............- ...................... .. 22
Regulations........................... .............................. 24
Fatty Acid Metabolism................. ........................ .25
Oaddation of Fatty Acids.... .......................... . ........ ' 26
Esterlfication of' Fatty Acids .......... . ....................... 27
_ Export of Triglyceride from Liver .............. . ............ ' .27
Summary ............................. .. ..................................... 32
MATERIALS AND METHODS ........................ ‘ ...................... ..36
Food Formulations .................... . ............ ....36
Blood Collections and Analysis ................ . .................. .....36
Urine Collections and Analysis................... .................... 37
Statistical Models ......................................................... 41

vii

RESULTS ............................................................................ 46

Intake and Energy Balance ............................................. 46
Non-esterified Fatty Acids ............................................. 47
Liver Fatty Acids .......................................................... 48
Body Condition Score and Body Weight ............................ 49
Milk Yield and Milk Components .................................... 50
Diseases ..................................................................... 50
Urine NMH and Creatinine ............................................. 51
DISCUSSION ...................................................................... 71
SUMMARY AND CONCLUSION ............................................ 78
LIST OF REFERENCES .......................................................... 79

viii

Table 1 .

Table 2.

Table 3.

Table 4.
Table 5.

Table 6.

Table 7.
Table 8.

Table 9.

LISLQLIABLES
Ingredients of the diets .................................................. 52'
Ls means for prepartum nutrient intake of the diets
(primiparous and multiparous cows)..............................‘...53

Dry matter and nutrient intake, and energy balance of cows

(Least square means) .................................................... 54
Ls means (ueq/Ll for non esterified fatty acids (NEFA) ....... 55
Ls means for liver fatty acids .......................................... 56
Ls means for body condition score (BCSI and body weight

(8W); Kg ................................................ 57
Ls means for milk yield and composition .......................... 58
Numbers of selected diseases peripartum ......................... 60

Mean for urine creatinine Imgldl) and N7-Methyl histidine

(nmollmll ...... ' ............................................. ' .............. s1

ix

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.

Figure 9.

W

DMI, primiparous cows ........................................... 62
DMI, multiparous cows ........................................... 63
Plasma NEFA- primiparous cows .............................. 64
Plasma NEFA- multiparous cows .............................. 65
BCS, primiparous cows ........................................... 66
BCS, multiparous cows ........................................... 67
8W - primiparous cows .................................. . ......... 68
BW - multiparous cows ........................................... 69
SCM yield of cows ................................................. 7O

The dry period for dairy cows is a lactation preparation period rather
than an insignificant rest period between lactations. During the dry period
a cow not only rests but the body is preparing for important changes that
will influence her next lactation. The mammary gland involutes, regenerates
and produces high quality colostrum. Two-thirds of fatal growth occurs
during the dry period and this growth takes priority over maintenance of the
cows own body tissues.

Plasma nonesterified fatty acid (NEFA) concentrations increase prior
to and at parturition, resulting in increased fatty acid uptake by the liver.
The fatty acids are esterified and stored as triglycerides in the liver.
Export of newly synthesized triglycerides from the liver as very low density
Iipoprotein occurs slowly in ruminants and is a major factor in the
development of fatty liver. Deposition of triglycerides in the liver, seems to
be a result of more uptake of NEFA than what can be oxidized and exported
(Herdt, 1988). Accummulation of triglycerides in the liver infiltrate liver
cells and interfere with its functions and causes some metabolic problems.

Therefore, nutritional strategies to minimize the elevation in plasma NEFA

1

2

prior to calving is expected to lower liver triglycerides at calving and may
decrease incidence of metabolic disorders le.g., ketosis, displaced
abomasum, retained placenta and milk fever). Research to determine
methods to reduce fatty acid delivery to the liver or to enhance hepatic

export of very low density Iipoprotein (VLDL) near calving is warranted.

Obiectiszes'

The general objectives of this study were:

1) To determine if a high energy and/or high protein diet prepartum
increases peripartum nutrient intake, improves nutrient balance as
indicated by plasma NEFA concentrations, affects protein turnover
(creatinine and 3- methyl-histidine ratio) and increases milk yield.

2) To determine the interrelationships of serum NEFA and prepartum

dietary energy density and protein content.

 

Marginal quality of feed, imbalanced rations, and inadequate housing all
characterize poor dry cow management practices. Deficient dry cow care
may result in decreased milk yield, increased incidence of periparturient
health disorders, and impaired fertility (Curtis et al., 1985). The result is
reduced overall milk production efficiency (pounds milk per unit cost).
Lactating cows need to be dried-off about 60 days before their expected
date of calving. Metabolically, the dry period is when a dairy cow must
alter her metabolic priorities for available nutrients from net tissue
deposition (e.g., mid-gestation) to reserve tissue mobilization (e.g., early
lactation) (Coppock, 1988). This metabolic transition does not occur
abruptly, but gradually over about a three week period prepartum.
Cessation of milking results in reabsorption of non-secreted milk, and rapid
loss of mammary secretory epithelial cells. The highest time for
susceptibility to new intramammary infections is the two weeks of

involution (Cousins et al., 1980; Neave et al., 1950). Once the udder

 

4

reaches a stable non-secretory state, susceptibility to new infection is
reduced (Nickerson. 1990). Susceptibility to new infections increases as
the udder starts to produce secretions in preparation for lactation in early

lactation (Cousins at al.. 1980;Neave et al.,1950;Nickerson. 1990).

A short dry period (< 40 days) reduces subsequent milk yield (
Coppock et al., 1974; Dias et al. 1982; Funk et al.. 1987; Schaeffer et al.,
1972). Also excessive days dry (> 70 days) have been shown to reduce
milk yield, which would decrease profit (Coppock et al., 1974; Dias et al.,
1982: Funk et al., 1987; Schaeffer et al; 1972). Factors such as age, milk
yield and days open affect optimum length of dry periods.

Inadequate days dry or poor quality dry cow nutrition may adversely
affect colostrum quality and quantity. Colostnrm is a milk-like fluid secreted
by mammary glands at the onset of lactation. Colostrum contains higher
concentrations of minerals (except potassium), proteins, fat, and fat soluble
vitamins (Naylor, 1986). It also contains high concentrations of maternal
immunoglobulin essential for passive transfer of immunity to the neonate.

Lipogenesis (fat deposition) occurs in mid-gestation, and lipolysis (fat
depletion) in late—gestation and early lactation (Bauman et al., 1980:
Coppock, 1988; Emery. 1988; McNamara et al. 1986: Vernon, 1988).
Fetal and placental maximum gain occurs in the last 11 weeks prepartum,

which places great burden on the dry cow (Prior et al., 1979) . Loss of

5 .
maternal nutrient reserve for fetal development can occur if nutrients are
inadequate. Presence of a second fetus increases pregnancy requirement.
which may cause the increased incidence of health disorders associated
with twin pregnancies (Koong et al.. 1982).

Dairy cows can metabolically adapt to the increased demands of a
given physiological state (i.e.. pregnancy. lactation and growth) by
homeorhetic regulation (Bauman et al., 1980; Emery; 1988). Increasing
Iipolytic activity is shown by the increased concentration of non-esterified '
fatty acids ( NEFA ) and B-hydroxybutyrate in the blood two to three weeks
prepartum (Gerloff et al.. 1986; Vernon, 1988). These metabolic changes
are associated with decreased insulin (i.e.. lipogenic hormone) activity
(Gerloff et al. 1 986; Vernon. 1988), and increased adipose tissue sensitivity
to the B-adrenergic agonist (i.e.. Iipolytic hormone_),e.g., epinephrine (Jaster
et al.. 1981: Vernon: 1988).

Lactogenesis is stimulated by the amount. and ratios of estrogen and
progesterone during late gestation (Erb. 1977). Milk yield depends on
secretory cell number (Knight et al., 1987; Tucker. 1987), and lactation
increases Iipolytic activity (Bauman et al.. 1980: Emery. 1988: McNamara
et al.. 1986; Vernon. 1988). Incidence of clinical or subclinical metabolic
disorders results from inability to adequately coordinate metabolism to meet

production demands.

Nutrient requirements for pregnancy represent nutrient amounts
necessary to support both growth rate and maintenance of fetus. placenta,
‘ uterus and mammary gland. Conceptus maintenance expenditure is a
significant portion of the total pregnancy requirement as evidenced by the
low efficiency of utilization for metabolizable energy (Ferrel at al., 1976;
Moe et al.. 1972).

Energy required by pregnant cows at term is 1 75% of that required
by an equal weight. of non-pregnant cows (Moe at al.. 1972). Concqms
nutrient requirements depend upon fetal numbers, rate of growth and birth
weight. Providing sufficient quantities of essential nutrients is just as
critical for. the dry cow as the lactating cow to maintain optimum
performance. Dry cow feeding programs with nutrient deficiencies (e.g.-
vitamin E. magnesium and selenium) have been associated with increased
health disorders in the periparturient period (Eger et al., 1 985; Hoffsis et al.,
1989).

Dietary recommendations for dry cow, have been established by the
National Research Council (NRC), for energy. protein, fiber, 14 minerals and
three vitamins (NRC, 1989). National Research Council (NRC) has
developed mathematical models of nutrient requirements for physiological
functions (i.e.. maintenance, growth. pregnancy. lactation. reserve

deposition and weight loss). These models are summed over all appropriate

 

7

states for a particular animal to determine the total nutrient requirement.
Increased energy above NRC recommendations during the dry period has
been shown in some studies to decrease metabolic and reproductive

disorders (Curtis et al. 1985).

Carbohydrate:

The amount of structural carbohydrates (i.e.. cell wall fiber) and non-
structural carbohydrates Ii.e.. cell contents) need to be balanced to meet
dietary energy density and maintain maximum dry matter Intake. Neutral
detergent fiber.(NDF), a measure of cell content, is negatively associated
with dry matter intake. Acid detergent fiber (ADF) is more associated with
feed digestibility than feed intake (Van et al.. 1985). A sufficient amount
of fiber needs to be maintained in the diet to optimize the balance of
carbohydrates and achieve maximum intake . Non-structural carbohydrate
levels need to be managed appropriately to obtain optimum body condition
of the dry cow and prevent over conditioning, which adversely affects

performance (Coppock. 1988; Smith et al.. 1981 ).

Protein:
Dietary protein is partitioned in relation to its rumen degradability and
solubility (NRC, 1989). Rumen-degradable and soluble protein support

microbial protein production. Rumen undegradable protein and microbial

protein supply the amino acids that support the animal productive function.
A greater percentage of dietary protein should be fed as soluble
protein. or non-protein nitrogen (NPN) if large amount of carbohydrates are
fed to dry cows. These protein sources are rapidly converted to ammonia,
an essential nutrient to cellulolytic bacteria (Sniffen et al., 1988).
Increased undegradable protein for dry cows has improved body
condition at calving and subsequent lactation performance in first lactation
heifers (Hook et al.. 1989; Van et al., 1989). A higher protein (15%) diet.
all from rumen-degradable sources, resulted in a 66% Incidence of downer
cow syndrome, compared to a lower protein (8%) diet (Julian et al., 1977).
Other studies showed reduced metabolic and reproductive health disorders
when feeding higher protein concentrations compared with NRC

requirements three weeks prepartum (Curtis et al., 1985).

NBQBecommendations:

National Research Council (NRC) incorporates a 10% activity
allowance of maintenance energy requirements for dairy cattle (NRC. 1 989).
This should be sufficient energy allotment to account for animal activity
when housed in stanchions with limited exercise or free stall facilities.
Additional increase in maintenance energy requirement of 10 to 20% for I
grazing quality or sparse pastures, respectively, are recommended (NRC.

1989). Nutrient requirements by the NRC are based on the animal being

9

exposed to stress free, thermoneutral environment (NRC, 1989). Climatic
factors (e.g., ambient temperatures, humidity, air movement and
precipitation), and animal factors (e.g., behavior, and hair length) all play a
role in modifying the range of thermoneutrality (Fox at al.. 1988; NRC.
1981). Heat stress during pregnancy reduced calf birth weight by 7 kg and
altered placental and maternal hormonal concentrations (Collier et al..
1982).

More milk is produced in the subsequent lactation from cows under
shade compared to ones without (Collier at al., 1982). Although this
difference in milk yield was not statistically significant. these researchers
conclude that, heat stress during pregnancy. may indirectly affect milk yield
through alterations in endocrine profiles, and reduced calf birth weights.

Ambient temperature below the lower critical temperature (i.e.. cold
stress) results in increased maintenance energy to maintain body
temperature (Young. 1983;). Increased dry matter intake usually
compensates for this increased energy need in marginal cold stress
situations (NRC. 1981 I. Environmental conditions need to be accounted for
in dry. cow diet formulation (NRC, 1981). Otherwise. extreme body
condition loss will occur under conditions of severe cold without dietary

adjustments, which will have a serious detrimental impact on subsequent

performance.

10

Dietary nutrient densities for dry cows. based on NRC
recommendations are determined on an assumed average dry matter intake
of 1 .6 to 2.0% of body weight (NRC, 1989).

If a cow consumes less dry matter than expected of diets containing those
suggested nutrient densities. she will ingest inadequate nutrient amounts to
meet her defined requirements. Over consumption of dry matter produces

the opposite results.

EeedJntake:

Feed intake of dry cows has been reported (Van Saun at al., 1989;
Zamet at al.. 1979) to range from 7 to 15 kg/d. and it Is affected by many
factors (NRC, 1987) (e.g., environmental and physiological). Much of the
intake variation is accounted for by animal parity and dietary forage:
concentrate ratio.

. Cows entering 'their first lactation consume less dry matter (mean, 7
to 12 kg I d) than older cows (Marquardt at al.. 1977; Van Seun at al.,
1989; Zamet et al., 1979) (mean 10 to 15 kg I d). Low forage diets are
consumed in lesser amounts than high forage diets (Coppock at al., 1972).

Decline of dry matter intake for dry cows beginning two to three
weeks prepartum, followed by a further drop 48 to 72 hours before calving,

was reported (Coppock etal., 1972; Marquardt et al., 1977; Van et al.,

1 989).

11

Management goals for dry cows should focus on maximizing dry
matter intake throughout the dry period to allow more rapid increases in
intake postpartum. This may be accomplished by feeding ad libitum a total
mixed ration appropriately formulated for energy and fiber levels.

Intake during the dry period (i.e.. close-up period) is critical in
preventing subsequent health disorders and increasing milk production
(Curtis at al., 1984; Curtis at al., 1985; Erb at al., 1988). Berlics at.
al. 1992 concluded that, liver triglyceride immediately after calving was
negatively correlated with dry matter intake. Nutrient imbalance (nutrient
deficiencies or excess) in the dry cow diet was reported to increase
incidence of parturient hypocalcemia (Curtis et al., 1984; Erb at al.. 1988),
hypomagnesemic tetany (Hoffsis et al.. 1989; Littledike et al., 1981),
retained placenta (Eger at al.. 1985) . mastitis (Erskine et al.. 1987), udder
edema (Emery et al.. 1969; Littledike at al.. 1981), ketosis (Foster, 1988:
Gerloff. 1988; Littledike at al. 1981), and displaced abomasum (Coppock
et al., 1972: Coppock. 1974). _ Epidemiologic studies have shown
periparturient health disorders not to be totally independent events. but a
complex of interrelated disorders (Curtis at al., 1983: Curtis et al.. 1985:
Erb et al.. 1981; Erb at al.. 1985: Erb at al.. 1988).

Forages should make up most of the dry cow ration. High forage
rations (>85%), have been thought to maintain maximal rumen fill(i.e..

volume), stimulate rumen motility, and allow healing of rumen wall lesions

12
resulting from high-grain lactation rations. Many forage and roughage
ingredients can be fed to the dry cow when rations are appropriately
formulated for energy. protein. fiber. and mineral concentrations. Dry cows
require long course fibrous material to stimulate rumination and saliva flow
to promote maximal fiber fermentation.

Body condition score (degrees of body fatness) should be evaluated
during the dry period. Body condition scoring grades cows by amount of
subcutaneous fat stores over the loin, pelvis, and tailhaad into five
categories ( Wildman at al., 1982). emaciated (1): thin (2); average (3); fat
(4); and obese (5) . Body condition scoring is an excellent cow monitor that
is easily quantified for evaluation of nutritional programs.

Body condition at calving plays an important role in determining
subsequent health (Fronk at al., 1980;), productive (Gamsworthy at al.,
1987). and reproductive (Ducker et al., 1984) performance. Moderate
body condition scoreI3-3.5) is essential to support milk production in early
lactation, and to initiate reproductive cycle. Under good management
practices fewer than 10% of the dry cows should havehcondition scores
over 4.0 or under 2.5 (Ferguson et al.. 1988). Either extreme in body
condition results in reduced milk yield (Gamsworthy at al., 1 987). increased

health disorders (Fronk et al., 1980). and impaired fertility (Butler et al..

1 989).

13

Body reserves to be utilized in early lactation start to build in the late
lactation when energy balance is positive. For this reason attention should
be placed to ensure adequate reserve at calving.

High-producing dairy cows have a tremendous metabolic challenge
during early lactation to provide adequate substrates for synthesis of large -
quantities of milk. Intake of energy-yielding nutrients from the diet usually
is less than energy output in milk. resulting in mobilization of body tissue to
supply the nutrient deficit. Most cows can adjust their metabolism to meet
this challenge; some cows. however, are unable to make the necessary
adjustments to maintain homeostasis. Metabolic diseases or disorders

occur as a result.

 

Lactation ketosis and fatty liver are two interrelated disorders of
energy metabolism. Estimates of the incidence of ketosis range from 2
to 15% (Baird, 1982: Littledike at al., 1981). Subclinical ketosis may
occur even more frequently. Cows with clinical ketosis usually have
fatty liver (Foster 1 988).

Ketosis results in decreased milk production, increased veterinary
expenses. and possibly decreased reproductive life (Baird. 1982; Schults,

1988). Association has been noted between fatty liver and increased

14
susceptibility to disease and reproductive problems (Gerloff et al. 1986;
Reid. 1982).

A protocol to induce ketosis and fatty liver experimentally has been
developed (Mills et al.. 1986; Veenhuizen et al.. 1991). The protocol
involves subjecting
cows in early lactation (10 to 14 d postpartum). to moderate feed
restriction. and a dietary source of ketone bodies (i.e., 1 ,3-butanediol), fatty

liverand ketosis develop gradually over a period of two to four weeks.

LactationJretosis:

Ketosis (i.e., acetonemia) is a metabolic disease of dairy cows in early
lactation, which is characterized by increased concentrations in blood of the
ketone bodies. i.e.. 3-hydroxybutyrate (BHBA). acetoacatate (AcAc) and
acetone.

Cows usually are affected within the first three to four weeks
postpartum, with older cows being more susceptible (Schultz, 1988).
Economic losses occur through decreased milk production. treatment
expenses, and possibly decreased productive lifetime (Baird, 1982). as
of primary ketosis (i.e., that which occurs as a primary disease, not as a
secondary complication to another disease or disorder) are somewhat
nonspecific. Signs may include decreased rumen activity, decreased

appetite, nervousness or deranged behavior, and decreased milk production. I

15

Ketone bodies are present in urine and milk. Presence of fever is an
indication of infection.

The two major changes in blood are increased concentration of
ketone bodies and decreased concentration of glucose. Other biochemical
changes in blood include increased concentrations of nonesterified fatty
acids (NEFA) and acetate. and decreased concentrations of insulin.
triglycerides. free and esterified cholesterol. and phospholipid. Cows with
subclinical ketosis may show no outward signs. but have increased
concentrations of NEFA, and BHBA, and decreased concentrations of
glucose in blood; subclinical ketosis may either remit or progress into clinical
ketosis.

The general biochemical progression toward ketosis is thought to be
as follows: production of glucose by the liver is inadequate to meet
demands of increasing milk synthesis, and concentration of glucose
decreases in blood. Insulin, with concentration already being low during
early lactation, may decrease further and allow increased mobilization of
NEFA and glycerol from adipose tissue. Uptake of NEFA by liver increases
production of ketone bodies and deposition of triglyceride in liver. and this
sometimes leads to clinical ketosis. During early lactation. most high-
producing cows will go through some degree of hypoglycemia, increased
mobilization of NEFA and production of ketone bodies without progressing

to ketosis. Baird (1977) stated that ketosis represents one extreme of the

16

“normal” continuum of glucose deficit and fatty acid mobilization in early

lactation.

EattyJiver:

Fatty liver (i.e., hepatic lipidosis) is a general term for accumulation
of lipid in the hepatocyte or parenchymal cells of the liver. It occurs in
many species during various conditions. Reviews of fatty liver in dairy
cows include those oflReid. 1 982; Roberts et al.. 1981: Herdt. 1988; Herdt
at al., 1982; Emery, 1979; Emery et al., 1992; and Vazquez-Anon at al.,
1994).

Triglyceride is the primary type of lipid that accumulates in liver
during the postpartum period in normal (Collins et al., 1980). or
overconditioned cows (Fronk at al., 1980). Hepatic triglyceride also
accumulates during fasting in lactating (Brumby at al.. 1975; Herdt et al.,
1983:) or non-lactating (Reid et al., 1977) cows, or during secondary
disorders such as displaced abomasum (Herdt at al.. 1983). Triglyceride
also accumulates in liver of ewes with pregnancy toxemia, or a combination
of fasting and administration of phlorizin and epinephrine (Herdt, 1988).
Content of cholesterol esters increased postpartum in fatty liver (Collins et
al., 1980), and during fasting in lactating cows (Brumby et al., 1975). No

increase in cholesterol ester was observed in non-lactating cows (Reid et al..

17

1977). Phospholipid content may be decreased (Herdt etal., 1983),
unchanged (Brumby etal., 1975: Collins et al.. 1980; Reid at al., 1977), or
increased (Fronk et al., 1980). Cows obese at calving were subject to a
complex of postpartum diseases known as fat cow syndrome or fatty liver
syndrome, which include development of severe fatty infiltration of the liver
(Henderson at al.. 1982).

Degree of fatty liver has been measured in biopsy samples by using
microscopic point-counting methods, chemical methods. or buoyant density
(Herdt, 1988). According to Gaal et al.. 1983: the three classifications of
degree of fatty liver are mild (0-2096 of cell volume or < 5% by weight as
triglyceride), moderate (20-40% of volume or 5-10% of weight as
triglyceride). or severe ( >40% of volume or > 10% of weight as
triglyceride). f

Occurrence of fatty liver is related. to the degree of mobilization of
body tissue after calving. Excessive tissue mobilization is indicated by
increased NEFA's concentration in plasma and loss of body weight and
condition score. Deposition of triglyceride in tissues such as liver, which
normally takes up and utilizes NEFA, seems to be a result of more uptake
of NEFA. than what can be oxidized or exported (Herdt, 1988).

Severe or clinical fatty liver is characterized by decreased responses
to treatments for other diseases or disorders (Herdt, 1988). Fatty liver was

reported to decrease reproductive efficiency in some studies (Reid, 1983:

18

Reid at al., 1979; Reid et al.. 1983) but not in others (Gerloff at al.. 1986).
Cows with fatty liver had decreased white cell counts in blood (Reid at al.,
1984), and a greater incidence of infectious diseases (Gerioff at al., 1986).
Cows with fatty liver also retained bacteria in the mammary gland for longer
periods after an experimental infection (Hill, 1985).

Study of the ultrastructure of hepatocytes from cows with severe
fatty liver revealed increased cell volume, decreased volume of rough
endoplasmic reticulum per cell. and evidence of mitochondrial damage (Reid
et al.. 1980). The latter two changes were reflected in vivo by decreased
albumin concentration, and increased activities of
mitochondrial enzymes in blood. Grohn and Lindbergl1985). observed a
decrease in the amount of Golgi apparatus in fatty liver from ketotic cows.
and an increase peroxisome In mildly ketotic cows, but a decrease in
peroxisome in severely ketotic cows.

In ruminants, the liver is the. major source of endogenous plasma
triglyceride. with intestinal denovo synthesis being of little consequence
(Pullen at al., 1988). Secretion of very-low-density Iipoprotein (VLDL) by
ruminant liver, however. is low when compared with that of non-ruminants
(Kleppe et al., 1988). Pullen‘ et al. (1988) found that the turnover rate of
the stored triglyceride pool in sheep liver was decreased when the size of
that pool increased. suggesting that fatty liver may further decrease the

ability to secrete Iipoprotein. Decreasing ability of hepatocytes to

19

synthesize apoprotein necessary for secretion of triglyceride rich Iipoprotein
was said to contribute to development of fatty liver (Herdt et al.. 1983;
Reid at al.. 1980). A

Some researchers have observed changes in concentrations of
lipoprotein in blood from cows with fatty liver. Concentrations of dextran
sulfate precipitable (DSP) Iipoprotein decreased in cows that developed
severe fatty liver in association with occurrence of displaced abomasum. but
they increased in cows that had fatty liver induced by fasting (Herdt et al.,
1983). In a field study. cows with naturally occurring fatty liver also had
decreased DSP lipids (Gerloff et al.. 1986).. Rayssiguier et al. (1988),
observed decreased concentrations of low-density lipoprotein (LDL) in blood
of cows with postparturient fatty liver.

Many investigators have attempted to identify metabolites or
enzymes in blood that would predict accurately the presence and degree of
fatty liver in dairy cows. These attempts have. for the most part. been
unsuccessful, and studies from different groups often provide conflicting
results.

In general, changes in blood associated with fatty liver include
increased concentrations of NEFA, BHBA, and bilirubin, and decreased
concentrations of glucose. total cholesterol. albumin. magnesium and insulin
(Reid et al., 1983). These changes are very similar to those that occur

during ketosis. Other changes noted in some studies include increased

20

activities of the enzyme aspartate aminotransferase (glutamate-oxaloacetate
aminotransferase) (Grohn etal., 1983; Herdt et al.. 1982; Lotthammer,
1982; Schultz, 1988), ornithinecarbamoyltransferase (Grohn et al.. 1983),
acid and alkaline phosphatase (Begin at al.. 1988), and glutamate
dehydrogenase (Begin at al.. 1988). Gerloff at al. (1986). noted a negative
correlation between concentrations of the thyroid hormones and degree of
fatty liver.

Now. liver biopsy remains a necessary procedure for accurately
determining content of lipid in liver (Herdt, 1988). It is obvious that
improved diagnostic techniques for fatty liver are needed.

Reid and Roberts (1982), and Gerloff at al. (1986 a, b, c) proposed
that fatty liver may be more an indicatorof negative energy balance during
early lactation than a separate disease. Furthermore, negative energy
balance may be the causative 'factor in increased susceptibility to other
diseases and reproductive problems. Although. the general cause of fatty
liver seems to be excessive uptake of NEFA by the liver, cellular and

molecular mechanisms of development remain to be detarrnined.

EaLQomSyndrome:
Morrow (1 976) and Morrow et al. (1 979) describe fat cow syndrome
as a complex of metabolic and infectious disorders, occurring during the

postpartum period in cows that calve in an obese condition, Signs of the

21

syndrome include anorexia, weakness, and associated diseases that may
include milk fever. ketosis, displaced abomasum, retained fetal membranes.
metritis. or mastitis. Response to treatment for these diseases usually is
poor; rates of mortality up to 25% have been observed in affected herds
(Marrow et al., 1979).

Cows with fat cow syndrome have fatty livers. Obesity at the
onset of lactation may lead to rapid mobilization of NEFA. which is
worsened by the decreased appetite usually observed in such cows (Bines
at al., 1983; Treacher et al., 1986). Biochemical changes in blood include
decreased concentration of triglycerides and increase concentrations of
NEFA. ketone bodies. urea, and bilirubin (Morrow, 1976). Concentration of
glucose may be either decreased or increased.

Increased mobilization of body tissue by fat.cows after parturition
was observed to be a combination of adipose tissue and muscle mass (Reid
et al., 1986). Reid at al.(1986) speculated that mobilized body protein was
not adequate for synthesis of lipoprotein. contributing to development of
fatty liver in the fat cows. Researchers should determine the role of quantity
and quality of dietary protein in preventing and alleviating fatty liver and fat

cow syndrome in early lactating dairy cows.

 

Because of ruminal fermentation of dietary carbohydrates, little
glucose is available for absorption from the small intestine of ruminants.
Ruminants are dependent on high rates of gluconaogenesis to supply their
needs for glucose. Use of large quantities of glucose by the mammary
gland of high-producing cows creates a tremendous demand for
gluconaogenesis most of which occurs in the liver. Glucose utilization and
gluconaogenesis have been reviewed (Lindsay, 1979; Young. 1977) and will

be discussed only briefly.

ErecursorsijEathways:

Precursors for gluconaogenesis include propionate, lactate, glycerol
and amino acids. Reynolds at al.( 1988 a,b,) measured maximal
contributions of propionate. lactate and amino acids to be 55. 18 and 16%
of net hepatic glucose production. Pyruvate and glycerol supplies less than
2% of glucose production in another study (Lomax et al.. 1983).
Propionate is the predominant gluconaogenic precursor in fed ruminants,

and gluconaogenesis increases with increased propionate supply.

22

23

About 50% of the lactate flux was derived from glucose in lactating
cows, with the remainder presumably being absorbed from the
gastrointestinal tract (Baird et al., 19833). Recycling of glucose through
lactate accounted for only 2% of glucose flux in lactating cows (Baird et al..
1983). Reynolds et al. (1988), determined that the net amount of lactate
taken up by the liver of early-lactating cows could provide a maximum of
18% of hepatic glucose production. Baird et al. (1983) concluded‘that.
most lactate taken up by the liver of lactating cows is oxidized or used for
synthesis of compounds other than glucose.

Glycerol may become quantitatively more important for
gluconaogenesis in COWS with severe negative energy balance during early
lactation (Feel at al., 1987). _The contribution of amino acids to
gluconaogenesis in ruminants has been debated. with estimates ranging
from 1 1 to 30% (Lindsay, 1973; Reynolds et al., 1988).

The pathways of glucose synthesis in ruminants are similar to those
in non-ruminants. Subcellular distribution of enzymes may vary. however.
Initial metabolism of propionate occurs in the mitochondria. Propionate is
activated to propionyl-CoA, carboxylated (to mathylmalonyl-CoA. and
converted to succinyl-CoA and then oxaloacetate (0AA).
Phosphoenolpyruvate carboxykinase (PEPCK), the major enzyme controlling
conversion of propionate to glucose, is found In both mitochondrial and

cytosolic compartments in ruminants (Ballard et al.. 1969). 'lhs.OAA

24

can be converted to phosphoenol pyruvate (PEP) in mitochondria. with PEP
exiting the mitochondria for conversion to glucose, or alternatively, OAA
can leave the mitochondria as malate. be reconverted to OAA in the cytosol,
and then converted to glucose. Lactate. pyruvate and amino acids that are
converted to pynrvate require pyruvate carboxylase (PC) to be converted to
OAA. Ruminants have PC located in both cytosolic and mitochondrial
compartments. Propionate. lactate. pyruvate and amino acids all require
participation of the citric acid cycle or at least are metabolized through
common intermediates.

Glycerol, on the other hand, enters the glyconeogenic pathway at the triose

phosphate stage, and thus its metabolism is independent of the citric acid

cycle.

Regulation:

Substrate availability is a primary regulator of gluconaogenesis in
ruminants; gluconaogenesis increases after eating and decreases with
fasting in ruminants, which are opposite the responses observed in non-
ruminants (Young,1977). Hormonal controls of gluconaogenesis in
ruminants have been summarized (Bassett, 1978; Trenkle: 1981).
Glucagon stimulates glycogenolysis and promotes gluconaogenesis,
whereas insulin largely exerts its effects on peripheral tissues to increase

glucose utilization. Glucocorticoid increases gluconaogenesis by increasing

25

delivery of amino acids to liver from skeletal muscle and increasing their
incorporation into glucose. Although growth hormone increases availability
of glucose for milk synthesis (Peel at al., 1987), direct effects on
gluconaogenesis have not been shown. Catecholamine and thyroxine

evidently stimulate gluconaogenesis in dairy cows.

Eatty_Acid_Metabelism:

Lipid metabolism in the liver of ruminants has been reviewed (Bell,
1980; Emery at al.. 1991). The liver of ruminants Is a major organ for
energy processing (Emery et al. 1992); which is similar to the situation in
non-ruminants. In contrast to many non-ruminant species, however. the
liver is quantitatively unimportant in fatty acid synthesis (Ballard at al.,
1969). Most synthesis of fatty acids in ruminants occurs in adipose tissue
(Vernon, 1980). Some synthesis of fatty acids from acetate occurs in
nrminant liver. Constant demand for gluconaogenesis in ruminants places
use of cytosolic OAA for lipoganesis at a disadvantage.

Fatty acids are taken up by liver in proportion to their blood flow and
concentration reaching the liver (Bell, 1980; Emery et al 1992).E m e r y
(1993) stated that. one-third of the circulating NEFA are received by the
liver, which is considered very high relative to the proportion of body weight
present as liver. Rate of uptake can be modified further by changes in the

NEFA to albumin ratio, with high ratios favoring increased uptake

26

(Bell.1980). The fractional extraction of NEFA from blood by liver is about
10% in sheep (Katz at al., 1969) and early-lactating dairy cows (Reynolds
at al., 1988). Major NEFA in ruminants are palmitic. stearic and oleic acids
(Bell, 1980), with stearic acid being utilized more poorly by the liver than
either palmitic or oleic acids. Fatty acids are toxic within cells and so are

activated quickly to acatyl-CoA esters and either oxidized or esterified (Bell.

1 980).

About 10% of the NEFA taken up by ruminant liver are oxidized to
CO, (Jesse et al.. 1986 a. b: Lomax et al., 1983). Mitochondria from
ruminant seems to oxidize NEFA at slower rates than mitochondria non-
ruminants (Koundakjian et al., 1970).

A major fate of NEFA within the liver may be conversion to the
ketone bodies. ACAA and BHBA. Hepatic ketogenesis in ruminants occurs
in mitochondria via the 3-hydroxy-3-methylglutaryl- CoA (HMG-CoA)
pathway. similar to non-ruminants (Bell. 1980). The limiting step for
oxidation and ketogenesis from NEFA is catalyzed by camitine
palmitoyltransferase l (CPTI) (Butler et al., 1988). which regulates entry of
NEFA into mitochondria. Ketogenesis thus depends to a great extent on
delivery of NEFAto the liver, which increases during starvation and ketosis.

Alternate viewpoints describe increased ketogenesis within

27

mitochondria as either an inhibition of the citric acid cycle's activity. or as
an over flow process in which citric acid cycle activity is unchanged (Ballard
at al., 1969). Zammit (1984) suggests that citric acid cycle activity is
regulated by NADINADH ratios so that total energy production is
maintained.

Although most circulating acetate originates from fermentation in the
reticulo-rumen and the lower gut. substantial production of endogenous
acetate occurs in the liver of cows (Lomax et al., 1983; Reynolds et al.,
1988; Snoswell et al., 1978land sheep (Bergman et al., 1971). The liver
may also utilize substantial quantities of acetate (Pethick et al., 1981).

Enzymes for esterification are located on the endoplasmic reticulum
and the outer mitochondrial membrane. Altemata pathways besides
esterification of glycerol-3-phosphate have been proposed (Benson et al.,
1971). Herdt et al., (1988) reported large increases in activity of
phosphatide phosphohydrolase early during development of induced fatty
liver in sheep, whereas activities of glycerol-phosphate acyltransferase and

diacylglycerol acyltransferase increased more slowly and to a lesser degree.

E I [I . l . I f l' _
ln non-ruminant animals, NEFAs are esterified in the liver to form

triglycerides. which then are packaged into VLDL and secreted (Havel,

28

1987). The capacity of ruminant liver to esterify fatty acids, however,
evidently is greater than its capacity to export triglyceride as VLDL.
Reasons for the apparently low capacity of. ruminant liver to synthesize
and/or secrete VLDL are not known. At least three hypotheses have been
proposed. First, sheep hepatocyte have a continuous basal lamina that may
physically limit secretion of large particles such as VLDL (Grub at al.. 1971 ).
Second. insufficient synthesis of apoprotien may limit rate of formation of
VLDL (Herdt et al., 1983; Reid et al.. 1980). Third. low rates of synthesis
of phospholipid or cholesterol may limit formation of VLDL Brumby et al.,
1975; Fronk et al., 1980; Herdt at al., 1983). The first hypothesis was
rejected by Pullen et al. (1988), who directly measured secretion of labelled
triglyceride from the liver after injection of a tracer fatty acid into a
mesenteric vein.

The rate of synthesis of triglyceride in liver determines the rate of
VLDL secretion in non-ruminants, and the supply of other components of
VLDL is thought to be regulated by the rate of triglyceride synthesis. It
seems that regulation of VLDL secretion in ruminant liver may be different
from in non-ruminants.

Triglyceride synthesized in excess of the amount that can be exported
as VLDL accumulates as lipid droplets and can results in fatty liver. Pullen
et al.(1988) found that hepatic triglyceride in sheep exited in two pools: 1)

A microsomal pool presumably associated with secretion of lipoprotein. and

29

2) a lipid droplets pool that is relatively inert. Turnover of the inert droplet
pool was slower than the microsomal pool and 80 to 90% of the hepatic
triglyceride was in the inert droplet pool. Pathways for removal of the
accumulated lipid droplets are unknown in ruminants. but in non-ruminants
very little transfer of droplet triglyceride to VLDL occurs; Rather. stored
triglyceride in lipid droplets must first be hydrolyzed by lysosomal acid lipase
(Debeer at al.. 1982). The fatty acids that are liberated then can be
oxidized or reesterified and secreted as VLDL.

Characteristics and metabolism of lipoprotein in ruminants have been
reviewed (Emery. 1979; Grummer et al., 1988; Palmquist 1976). Bovine
Iipoprotein were isolated predominantly in the high-density lipoprotein (HDL)
range (90%), with lesser amounts of LDL (10%) and VLDL or chylomicron
(< 1 %). Apoprotein composition of bovine Iipoprotein differs from that of
non-ruminants (Grummer at al.. 1987).

Metabolically, the VLDL and chylomicron fractions. are the most
active in providing triglyceride-fatty acids to peripheral tissues. most of
which will be taken up by the mammary gland in lactating cows (Palmquist
et al.. 1978).

With onset of lactation. metabolism of adipose tissue is redirected toward
providing fatty acids to other organs of the body (Vernon. 1980). Lipolysis
increases and lipogenesis ceases (McNamara at al., 1 986; Pike et al., 1980;

Smith at al., 1988). These changes were shown to begin during the last

3O

requirements for the dry cow are the sum for maintenance. pregna month
before parturition. The result of changes in adipose tissue metabolism
during early lactation is a tremendous increase in the ability of adipose
tissue to mobilize NEFA.

As discussed already, NEFAs are taken up in large quantities by liver.
kidney, heart and skeletal muscle (Lindsay. 1975). It is likely that the
mammary gland also takes up NEFA directly when concentrations of NEFA
are increased in blood (Pullen at al 1984). '

Peripheral utilization of ketone bodies by ruminants has been
reviewed (Heitman et al., 1987.). Although most production of ketone is by
gut mucosa and liver as discussed already, there is evidence for production
in skeletal muscle (Pethick 'et al., 1983). Unlike non-ruminants, brain and
nervous tissue of ruminant animals do not use ketone bodies. even during
starvation (Linsay et al., 1 976). Ketone bodies are used, however, by heart,
skeletal muscles. kidneys. and the lactating mammary gland (Heitman et al..
1987).

It has been assumed on the basis of early evidence (Bergman. 1971),
that bovine ketosis is a problem of overproduction of ketone bodies. and
that ketone body utilization is not limiting. Herdt (1988) suggested that
utilization may be impaired at high concentrations. Utilization also may be
decreased during deficiency of insulin (Balasse et al., 1971).

Ketone bodies exert several effects on metabolism and serve as

31

energy sources. Ketone bodies decrease proteolysis in skeletal muscle. and
serve as primers for fatty acid synthesis.

In addition ketone bodies may decrease gluconaogenesis in niminants
(Radcliffe et al.. 1983). Ketone bodies directly inhibit lipolysis in bovine
adipose tissue (Metz et al., 1972), and stimulate secretion of insulin, which

also acts to decrease lipolysis (Heitman at al.. 1987; Gerloff at al., 1986).

Nutrient my and reserve replenishment needs with additional requirements
for growth during the first two pregnancies. Maintenance energy
requirements can be dramatically increased by level of activity and adverse
environmental conditions. A wide variety of feed ingredients can be
successfully fed to dry cows as long as rations are appropriately formulated
to meet energy, protein, minerals and vitamins requirements.

Dry matter intake declines as a cow approaches calving. therefore. dietary
nutrient density needs to be adjusted to compensate. This suggests that
a single dry period diet may be inappropriate to sufficiently meet
requirements throughout the dry period. A close-up cow group may be
important. especially for herds experiencing increased incidence of health
disorders around calving.

This second dry cow group would also ease monitoring of cows for
impending parturition. particularly those with uncertain breeding dates and
metabolic disorders. The early dry cow ration is formulated for high fiber

and low energy density while the close-up ration contains higher energy

density

32

33

with less fiber. Both rations contain sufficient other nutrients based on
expected intake. This two group system provides maximal flexibility in
managing for optimum body condition at calving.

A good dry cow program should result in reduced incidence of metabolic
diseases. complete pregnancy with available calf and maximize genetic

potential for milk production. Overall a dry cow program is critical to

performance.

 

Eighty Holstein cows were used (n=80). forty primiparous (first
lactation) and forty multiparous (had one lactation or more). in a complete
randomized block design (CRBD). The cows were housed in MSU dairy barn
and fed a dietlTMR) formulated according to NRC recommendations. Four
weeks before the projected calving date. the experimental period began and
continued until calving. Water was available 24 h a day. Cows entered the
experiment in blocks of four, based on projected calving dates with
primiparous and multiparous cows in separate blocks (20 blocks). Cows
were fed ad libitum at about 1500 h. each day. one of four experimental
diets, from four weeks prepartum until calving. Orts were measured at
about 1300 h. The four diets contained:

1) Low energy and low protein (1 .3 Mcal NEI/Kg and 12.2% crude

protein (CP).

2) Medium energy and medium protein (1.49 Mcal NEl/Kg and 14.2%

crude protein (CP).

3) Medium energy and high protein (undergradable intake protein

added). (1.48 Mcal NEllKg and 16.2% CPI.

34

35
4) High energy and high protein (1.61 Mcal NEI lKg and 15.9% crude
protein (CPI.
* Diets and predicted intake are described in table 1 and 2.

After calving all cows were fed ad libitum according to NRC
recommendations twice daily (0330 h and 1600 h). until week 10
postpartum.

Starting six weeks before the projected calving data, feed intake was
recorded daily. Cows were weighed 2xlweek (Thursday and Friday) at
0800 h every week until the fourth .week postpartum, and then 2x I week
every other week until the end of the experiment (10 weeks postpartum).
Blood samples (for NEFA concentrations). as indicator for nutrient status.
were collected via puncture of the tail vein 2xlweek (Monday and Thursday)
at about 1 600 h. and daily starting 10 days before expected date of calving
until 7 days postpartum. Milk yield was recorded daily (3x I d) and milk
composition measured 2x I weekIMon. and Thr.). Fat. protein & lactose in
milk were measured using an infrared analyzer (Multispac, Wheldrake. UK)
at Michigan DHIA (East Lansing). Body condition score (1-5) was measured
at two week intervals by three different people, who didn't know the
treatments, and their scores were averaged. Urine samples for creatinine
& 3omethyl-histidine concentrations as indicator of muscle mass and protein
turnover. were collected 2x I week (Monday and Friday) at about 0700 h.

until week 4 after calving & than every other week until the and of the

36

experiment.

A liver (25-50 mg) biopsy was taken at 24 h postpartum using a
needle biopsy procedure. Health problems were recorded.
Eeedionnulations;

Cows were housed in a free stall until 6 weeks prepartum & then
moved to tie stalls until the end of the experiment. All cows were fed the
low diet ad libitumlTMR) from 7 weeks prepartum until 4 weeks prepartum
when the experimental diet started. Cows that were fed the high diet were
fed the medium diet for two days before they started the experimental diet,
in order to make a gradual shift from the low to' the high energy diets. Total
mixed ration (TMR) was fed throughout the trial ( table 1 and 2 I. Silage
dry matter was used to determine the amounts to be mixed to get the
required energy 8: protein concentrations.

Feed samples for the experimental dietslsamples of individual forages

and other ingredients) were taken weekly for analysis of dry matter, CP,

NDF, ADF. and Ash.

Bl ICII I' BIIEEEA l"
Samples were collected in vacutainer tubes containing EDTA to
prevent blood clotting. Samples were centrifuged the same day for 1 5 min.

at 3000 rpm and 4° C, and plasma was then collected and frozen at -20° C

until assayed.

37

A few months later 19 samples from. around day -35; -28; -21: -14;
-10; -7; -4; -2: -1: -0; 1: 2; 4; 7; 14; 28; 42; 56: and 70. were used for
NEFA analysis. Blood plasma samples were thawed overnight in the cold
room (4" CI. and 5 ul from each sampleltriplicate) were pipetted into a 96-
well microtiter plate. NEFA concentration was measured using NEFA-C
Kit(Wako chemicals USA. Dallas. TX); as modified by McCutchaon and
Bauman, 1986). in which 100 microliters from color reagent A were added
and shaken in the biolog machineIBiolog. lnc.. Hayward CA). After 30
minutes. 200 microliter of color reagent 8 were added and shaken in the

biolog. Thirty minutes later optical density was read at 550 nm wave

length.

Samples were collected in 50 ml tubes. by rubbing the vulva until the
cow urinate. About 5 ml from each sample was frozen (-20° C) for
later analysis.

Urine samples from weeks -2; -1; + 1; +2; and +3 weeks were
thawed and centrifuged at 3000 rpm for 15 min. and 4° C to remove
sediments. Five microliters were taken for creatinine analysis and the rest
was refrozen for N-methyl histidine (NT-MH) analysis. Raw urine samples

(not deproteinized) were analyzed for creatinine using Sigma Kit number

38

555-A (Sigma chemical CO., St. Louis,Mo) based on the Jaffe reaction.

The procedure for N'-MH analysis was reported by Simmons (1993).
Samples for NI-MH analysis were thawed and deproteinized with 50%
sulfosalicylic acid (SSA): (0.9 ml urine & 0.1 ml 50% SSA). The samples
were vortexed and centrifuged at 15000 rpm for 15 min. and 4° C. The
supernatant was diluted 1:1 with sodium hexanesulfonate because this
served as the mobile phase in the chromatograph. The amount of sodium
hexane sulfonic acid needed for dilution was based on the size of the peaks
that were eluted after different dilutions of the samples.

After dilution the mixture was vortexed and placed into a high-
performance liquid chromatography (HPLC) vial. The samples were chilled
at 4° C and then analyzed by reversed-phase HPLC separation using ion-
‘ pairing and post-column derivatization with o-phthaldehyda and fluorescence
detection (Friedman and Smith, 1980), NT-MH peaks in the samples were
identified on the bases of retention time as compared with NI-MH
standards. Concentrations of NI-MH in samples were calculated by
comparing the peak area of NI-MH in standards (50 mgIml) and samples.

A stock solution of 5 mM sodium hexanesulfonate with a pH 3.2 was
used as the mobile phase in the chromatograph. Sodium hexanesulfonate
increases the retention of and allows for good resolution of NT-MH. The
solution consisted of 1 L of HPLC-quality water and 0.94 g 1-hexane

sulfonic acid, and sodium salt (Aldrich, Milwaukee. WI; F.W. 188.22. 98%).

39

Approximately 1 ml glacial acetic acid was added dropwise to lower the pH
to 3.2. The solution was stirred for 30 to 45 minutes and then it was
allowed to stand for 30 to 45 minutes. Next. the solution was filtered
through a 0.45 um aqueous filter (47 mm diameter; Millipore Corp..
Bedford, MA) with a glass prefilter (Whatman GFIC, 4.25 cm; Whatman.
lnc., Clifton, NJ) on top of a Millipore-type all glass vacuum filtration system
with a 1 L flask. Lastly, a vacuum was applied for about one minute to
degas the solution. The solution was stirred rapidly with a magnet while it
was being degassed.

The o-phthaladehyde reagent was prepared by dissolving 30 g of
boric acid in 1 L of HPLC-quality H20. To adjust pH, 20 g of potassium
hydroxide was added to the solution first. than approximately 5 g of
potassium hydroxide was added slowly until a pH of 10.4 was achieved.
The solution was stirred for at least 30 minutes and than it was allowed to
stand for at least 30 minutes. Next, the solution was filtered in the same
manner as the mobile phase; however. the solution was not degassed.
Separately, a solution which contained 600 mg fluoraldehyde (Pierce OPA
crystals, catalog no. 26015; Pierce chemical CO.. Rockford, IL), 10 ml
ethanol. 200 ml of B-mercaptoethanol. and 1 ml of 30% (WN) aqueous
solution of Brij 35 (Pierce chemical CO.. Rockford, IL) was prepared. The
o-phthalaldehyde solution and the borate sclution was mixed in a dark glass

bottle just before it was used and stored under N2 gas. The borate solution

40
was prepared one day ahead of time. but the 0-phthaldehyde solution was
prepared just prior to use.

Injections of 25 ml of the samples were added to a 25cm by 4.6 mm
internal diameter. Vydac C-1 8 peptide/protein column (Rainin Instrument Co.
Inc., Wodurn, MA). The flow rate of the mobile phase was 0.8 mllmin and
isocratic conditions were used to separate NI-MH from other urinary ,
components. The column was periodically washed with acetonitrile to

remove non-polar contaminants which were retained on the column.

Clll' IIIIIIIIII Ill .1 II' _

1) nmoles NMH standard injected = ng NMH injected * 1 nmolel169.2 ng

2) nmoles NMH/injection = sample NMH area * nmoles NMH standard
injected/standard NMH area

3) nmoles NMH/ml urine = nmoles NMH/injection * 1 injectionlul sample

i’ 1 000 ul/ml

41
StatisticaLModels
The variations between treatments among different parameters from

the chosen independent variables was evaluated using the generalized
linearmodel procedure of (SAS, 1995) in the following models:

Umfiat .
Y... =p +c:r.+B,m +v.+ av... + Em,
Y... = Observed response for different liver
fat parameters.
p = Overall mean.
a. = Fixed effect of ith parity level .
Bin) = Random effect of jth block level within
ith parity level (to test parity).
y" = Fixed effect of kth treatment level .
Earn = Random residual effect. I
BodyJMeight:
Y." =p+q+§n +y+cw+yfi +.6+
a6. +6y. + aye“ + Em,
YukI = Observed response for body weight.
[I = Overall mean.
a. = Fixed effect of jth parity level.
8,“, = Random effect of jth block level within

ith parity level (to test parity).

Fixed effect of kth treatment level.

.75
II

42

(5. = Fixed effect of lth week level.
Emu = Random residual effect.
BodLCnnditianjcareJBCSI:

Y... =p+a.+%+yk+ay.k "I'Eum

Y“ = Observed response for BCS.

p = Overall mean.

a. =' Fixed effect of ith parity level .

BinI = Random effect of jth block level
within ith parity level (to test
parity).

)4. = Fixed effect of kth treatment level .

Emu Random residual effect.

[I I .fi If H 'l (NEE!)-

Yru =p+q+fi,, +y+qy+yg +.6+
a6. +6y. + GYOM + Em»

Yijkl = Observed response for NEFA.

p = Overall mean.

a. = Fixed effect of jth parity level.

81m = Random effect of jth block level within
ith parity level (to test parity).

y. = Fixed effect of kth treatment level.

43

6. = Fixed effect of lth day level.
E..." = Random residual effec.
M'll Illll'll c I .

Y... =p+q+§. +.y+q.v+y3§ 4.6+
a6. +6y.. + aye... + E“...

Y... = Observed response for milk yield and
components.

p = Overall mean.

a. = Fixed effect of jth parity level.

8..., = Random effect of jth block level within
ith parity level to test parity).

yk = Fixed effect of kth treatment level.

6. = Fixed effect of lth week level.

E"... = Random residual effec.

Dn/_MatteLlntake:

Y... =p+q+B. +y+qy+y§ +.6+
a6. +6y... + ova... + E...”

Y... = Observed response for dry matter intake.

[1 = Overall mean.

a. = Fixed effect of jth parity level.

8..., = Random effect of jth block level within ith

44

parity level (to test parity).
y. = Fixed effect of kth treatment level.
(5. = Fixed effect of lth weak level.

E..." = Random residual effec.
Energy_Balance:

Y... =p+q +.y+or.y +5., +.6+q6+§y+
cryfi... + E...”

Y... = Observed response for energy balance.

p = Overall mean.

or. = Fixed effect of jth parity level.

C...” = Random effect of cows within ith parity and
kth treatment.

y. = Fixed effect of kth treatment level.

(5. = Fixed effect of lth week level.

Erma: Random residual effect.

45
Eermulas_used_fnLenergy_balance_(NBC._1989L(McaLleI:
Energy for milk production = Milk yield * 2.2 ( 41.63 * fat +

24.13 * protein +21.6 *
lactose-11.7ZII1000 ‘

Energy for maintenance :

Lactating cows 0.08 * BW-°-7‘

Dry cows 0.104 * BW'M'5

Intake_and_Energy_Balance:

No differences in DMI were observed among the different treatments
prepartum (P > 0.58; Table 3) or postpartum (P > 0.7). However there
were differences in intake of CP, NDF and NEI (P < 0.05) and that is
because the diets were formulated to obtain such differences. Prepartum
feed intake was higher for multiparous cows than for primiparous cows (P
< 0.01 ). There was a less decline in the dry matter intake in the last three
weeks before calving for primiparous cows(13%) than for multiparous
cows(18%: P<0.01; Figure 1 and 2). Dry matter intake increased about
53% for primiparous cows from week 1 to weak 9 postpartum. and about
72% for multiparous cows (P<0.01).

Data from week 4 prepartum and week 10 postpartum for energy
balance were dropped because of missing values. Energy balance increased '
prepartum as dietary energy density increased '(P < 0.02). and all cows
were in positive energy balance prepartum except the primiparous cows fed
the low diet. Multiparous cows had higher energy balance than primiparous
cows prepartum (P < 0.01), but there was no difference postpartum (P >
0.2). All cows were in negative

46

47

energy balance in the first 10 weeks postpartum. Dietary treatments did
not affect energy balance postpartum (P > 0.4). There was a tendency for
the calculated energy balance of primiparous cows to drop more rapidly
toward calving (83%) than it did for the multiparous cows (57%). Le.

parity’week interaction (P < 0.1).

Prepartum NEFA concentrations were lower for multiparous cows than
for primiparous cows. and the reverse is true postpartum (P < 0.05). The
concentrations of NEFA increased from 90 pquL at 4 weeks prepartum to
about 1000 peq/L around calving for multiparous cows (Figure 4), and 800
pquL for primiparous cows (Figure 3; P < 0.01). The concentrations
dropped after calving until they became stable at 4 weeks postpartum,
which was the same as 4 weeks prepartum. Dietary treatments did not
affect NEFA concentrations prepartum for primiparous cows (P > 0.67;
Table 4). but there was a difference prepartum in NEFA concentration for
multiparous cows due to treatments (P < 0.01 I. The cows that were fed
the L diets had higher blood NEFA concentrations than that which were fed
the H diets. Multiparous cows tend to mobilize more NEFA before
calving than primiparous cows i.e. parity'day interaction (P < 0.05).
Multiparous cows receiving different dietary treatmmts responded

differently with respect to NEFA concentrations during prepartum period.

48

The cows fed L and M diets had more NEFA than the cows fed H and MH
diets interaction (P < 0.01). Multiparous cows fed L diet prepartum had
more NEFA than cows fed H diet (P < 0.01). Also multiparous cows fed
M diet had higher NEFA than cows fed MH diets (P < 0.05).

Postpartum NEFA concentrations for primiparous cows were not
affected by dietary treatments (P > 0.62), and the same results were

obtained from the multiparous cows (P > 0.43).

LirLeLEatnLAcid:

Liver fatty acid results are shown in table 5. Liver fatty acid
concentrations (mg/g wet liver) at calving were higher for multiparous than
for primiparous cows (P < 0.05). Dietary treatments did not affect liver
fatty acid concentrations for primiparous (P > 0.45) or multiparous cows
(P > 0.19), also they did not affect triglyceride fatty acid per gram liver for
primiparous cows (P > 0.42) or multiparous cows (P > 0.16).

There was a treatment effect (P< 0.07) on liver fatty acid and liver TG
when we combined the multiparous and primiparous cows. in which the (LL)
diets were higher than the (HH) diets (P<0.02). Diets also had no effect
on liver fatty acid percentages for primiparous (P > 0.1) or multiparous
cows (P > 0.91). However, the numerical values for liver fatty acid

contents tend to decrease with the increase of energy density.

49

Body condition scores were adjusted using BCS 4 weeks prepartum
as covariate. and the first block from each parity was taken away from the
statistical analysis because of several missing values. Body condition score
was higher prepartum for multiparous cows than primiparous cows (P <
0.01; Table 6). and there was no difference postpartum (P > 0.34). Body
condition scores decreased from around 3.25 at 4 weeks prepartum to
around 2.25 at 10 weeks postpartum (Figure 5 and 6). There were no
differences among treatments prepartum (P > 0.51) or postpartum (P >
0.71 I.

Dietary treatments did not affect 8W prepartum (P > 0.12) or
postpartum (P > 0.12). but they affected changes in 8W for multiparous
cows. Multiparous cows had higher 8W than primiparous cows (P < 0.01).
Body weights increased about 7% from 4 weeks prepartum to calving (P <
0.01). and then dropped sharply after calving until about two weeks
postpartum after which they remained fairly stable (Figure 7 and 8).
Multiparous cows lost more weight postpartum than primiparous cows fed
the same diets i.e. wk‘Parity interaction (P < 0.01). As crude protein
Increased in the diet for multiparous cows before calving, they gain more

weight before calving (P<0.05) and lost more weight after calving (P <

0.05).

50

There was no effect of different treatments on milk yield for
primiparous cows (P > 0.28; Table 7) and multiparous cows (P > 0.99).

Milk yield was higher for multiparous cows than primiparous cows (P
< 0.01). Milk yield increased with time after calving and reached the
maximum in about nine weeks (P<0.01; Figure 9). Multiparous cows
reached the maximum milk production (6 wk) before primiparous cows (10
wk) i.e. parity‘week interaction (P<0.01). There was no difference

between the different treatments on milk components.

Diseases
Incidence of milk fever. ketosis, displaced abomasum. metritis. clinical
mastitis and udder edema has been summarized in Table 8. The prepartum

diets did not significantly affect the incidence of peripartum diseases (P >

0.2).

51

.V. . “LII.” ID I' . _

The urinary concentration of creatinine for primiparous cows were
higher than for multiparous cows (Table 9: P<0.08). The different
treatment had no effect on urinary NT-MH, creatinine. and their ratios
(P>0.1), except for the ratios prepartum for primiparous cows (P<0.01)
and multiparous cows (P<0.06). The H and MH diets had higher ratios
than the L and M diets respectively. The decrease in urinary creatinine
concentrations from 2 wks prepartum to 3 wks postpartum was about 47%
for the primiparous cows. Urinary NT-MH to creatinine ratios dropped about
4% for the primiparous cows ), and 24% for multiparous cows (Fig. 11),
from 2 wks to 1 wk prepartum. and then increased about 46% for
primiparous and 73% for multiparous cows at 1 wk postpartum. and then

dropped again about 21% for primiparous and 32% for multiparous cows

at 3 wks postpartum.

APPENDICES

52
APPENDIX A

Table 1. Ingredients of The Diets

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment
Ingredients LL MM MH HH
%DM
Alfalfa Silage 31 .4 32.9 32.2 33.2
Corn Silage 30.3 20.9 20.6 1 1 .8
Cotton Seed 30.0 15.4 15.7 1 .8
Hulls
Corn Grain 0.0 0.0 0.0 18.0
Cracked
Corn Grain 5.4 23.4 20.0 23.2
Ground
Soybean Meal 0.9 5.5 1.4 10.0
44
Soyplus 0.0 0.0 7.4 0.0
Anion Salts 1 .3 1.3 1 .3 1 .3
Ca17 P21 0.3 0.1 0.1 0.0
Lime 0.0 0.1 0.1 0.3
Blood Meal 0.0 0.0 0.8 0.0
MineraINitamin 0.4 0.4 0.4 0.4

 

 

 

 

 

LL = Low energy, low protein diet.

MH
HH

= Medium energy. medium protein diet.
= Medium energy, high protein diet.

High energy, high protein diet.

 

53

APPENDIX B

Table 2. Ls MEans For Prepartum Nurtrient Intake Of The Diets
(Primiparous and Multiparous cows)

 

 

 

 

 

 

 

 

 

 

 

 

Treatment
Nutrient Intake LL MM MH HH
DM. Kgld 11 .5 12.2 12.5 13.0
CF. Kgld 1.4 1 .7 2.0 2.1
NEL. Meal/d . 14.9 18.2 18.5 21.2
NDF, Kgld 5.9 4.9 5.0 3.8
ASH. Kgld 1.0 1.0 1.1 1.1

 

 

LL = Low energy, low protein diet

MM = Medium energy. medium protein diet.
MH = Medium energy, high protein diet.
HH = High energy, high protein diet.

54

APPENDIX C
Table 3. Dry matter and nutrient intake. and energy balance of cows.
(Least square means)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Energy balance units (Mcal NE./d)

 

 

 

 

Treatments

ll. MM MH HH SEM P
No.of cows 1 0 1 0 1 0 1 0
DM (Kgld)
Primiparous 9.2 1 0 10.5 1 0.3 1 .0 0.67
Multiparous 1 3.8 1 3.8 14.4 15.7 1 .2 0.58
CP (Kgld)
Primiparous 1 . 1 1 .5 1 .7 1 .7 0.20 0.02
Multiparous 1.7 2.0 2.3 2.5 0.2 0.01
NDF (Kgld)
Primiparous 4.7 4.3 4.2 3.0 0.4 0.04
Multiparous 7.. 1 5.5 5.9 4.8 0.01 0.01
NEL (Mcal/dI
Primiparous 1 1.9 15.9 15.7 16.3 1 .5 0.09
Multiparous 1 8.0 20.5 21 .3 25.2 1 .8 0.02
Energy balance
3wksprepartum
Primiparous -1.3 2.9 2. 1 4.4 1 .4 0.02 .
Multiparous 2.7 4.8 6. 1 1 0.3 1 .4 0.01
Energy balance
9wks postpartum
Primiparous -2.5 -0.9 -0.4 -2.0 0.9 0.4
Multiparous -1 .3 -2.4 -4.4 -1 .4 1 .4 0.4

 

55
APPENDIX D

Table 4. Least squares means (ueqlLIfor nonesterified fatty acid (NEFA)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatments

LL MM MH HH SEM P
No. of coWs 10 10 10 10
4 wks
prepartum to
calving
Primparous 258 285 266 220 32.6 0.7
Multiparous1 202 ' 221 166 134 14.9 0.01
2 wks
prepartum to
calving
Primparous 307 349 322 266 45.1 0.73
Multiparous1 243 274 1 94 148 21 .5 0.01
Calving to 10
wks postpartum
Primparous 309 301 307 347 25.5 0.62
Multiparous 415 429 459 517 45.1 0.43
Calving to 2
wks pospartum
Primparous 422 413 410 478 42.5 0.71
Multiparous 595 595 636 725 70.2 0.56

 

 

 

 

 

‘ Contrast LL vs HH(P < 0.01). and MM vs MH (P < 0.05)

 

56

APPENDIX E

Table 5. Least squares means for liver fatty acids

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatments

LL MM MH HH SEM P
No. of cows 8 6 9 7
mg of total fatty
acids per 9 liver
Primparous 26.63 18.61 20.12 18.91 4.2 0.48
Multiparous! 40.1 34.3 31.0 27.5 4.7 0.23
mg of triglyceride
fatty acids per 9
liver
Primparous 10.22 7.5 8.85 5.63 2.1 0.42
Multiparous' 20.15 22.7 14.4 13.4 3.8 0.17
% fatty acids in
T6 per total fatty
acids in liver
Primparous 33.76 39.0 41.22 27.29 4.3 0.1
Multiparous 52.3 50.4 46.3 46.0 4.9 0.90

 

 

 

 

 

 

 

No. of cows for multiparous. LL = 9; MM =

e;MH== 9; HH = 7

 

 

 

 

 

57

APPENDIX F

Table 6. Least squares means for body condition score (BCSI‘and body

weight (BWI: kg

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatments

IL M M M H SEM P
No. of cows 9 9 9 9
BCS 1 wk
prepartum (cov -
4 wk)
Primparous 3.35 3.33 3.43 3.39 0.1 0.64
Multiparous1 3.49 3.50 3.50 3.63 0.1 0.51
BW last month
prepartum
Primparous 634 602 628 584 16.9 0.12
Multiparous‘ 755 756 750 726 19.7 0.62
8W first 10 wks
postpartum
Primparous 634 602 628 584 16.9 0.12
Multiparous1 648 649 644 635 17.4 0.94
BCS wk 5 to 10
postpartum (cov
-4 wk) .
Primparous 2.46 2.49 2.61 2.50 0.1 0.73
Multiparous 2.30 2.32 2.36 2.43 0.2 0.71

 

‘ BCS is a five point scale (1 = extremely thin, to 5 = overconditionned)

 

58

APPENDIX G

Table 7. Least squares means for milk yield and composition

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatments

L M MH H SEM P
No. of cows 10 10 10 10
Milk Yield
( kgldI
Primparous 35.4 32.1 31 .5 32.9 3.2 0.28
Multiparous1 45.4 45.5 45.0 44.8 3.4 0.99
SCM (Kgld)
Primparous 32.5 30.2 30.0 31 .1 2.5 0.39
Multiparous1 42.1 43.5 42.6 43.3 2.8 0.88
Fat% 3.5 3.7 3.7 3.7 0.1 0.57
Primparous
Fat% 3.6 3.8 3.7 3.8 0.1 0.40
Multiparous1
Fat (Kgld)
Primparous 1 .2 1 .2 1.1 1.2 0.1 0.80
Multiparous 1 .6 1 .7 1 .6 1 .7 0.1 0.70
Protein %
Primparous 3.0 3.0 3.1 3.1 0.1 0.62
Multiparous 3.1 3.1 3.0 3.2 0.1 0.08
Protein (Kgld) I
Primparous 1 .0 1 .0 1 .0 1 .0 0.1 0.26
Multiparous 1 .4 1 .4 1 .4 1 .4 0. 1 0.81
Primparous 5.0 5.0 4.9 4.9 0.04 0.82

 

 

 

 

 

 

 

 

Table 7. (Cont’d)

59

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lactose %

Primiparous 5.0 5.0 4.9 4.9 0.04 0.82
Multiparous 4.9 4.9 5.0 5.0 0. 1 0.51
Lactose (Kgld)

Primiparous 1 .8 1 .6 1 .6 1 .6 0.2 0. 1 9
Multiparous 2.2 2.3 2.3 2.2 0.2 0.99
SCC 1 23 1 1 2 1 86 204 40 0.31
Primiparous

SCC 1 83 246 245 1 80 74 0.86
Multiparous

 

‘ Contrast L vs H, and M vs MH P< 0.07

 

60

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61
APPENDIX I

Table 9. Mean for Urine Creatinine (mgldl) and N’Methylhistidine (nmols/mll

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment
Item ' L M MH H SEM P
Creatinine
Prepartum
Primiparous 1 95 1 92 1 86 1 98 27 0.9
Multiparous 1 71 169 1 69 1 66 16 0.9
NT-MH Prepartum
Primiparous 21 4 234 264 289 30 0.4
Multiparous 1 65 238 1 95 222 40 0. 1
NT-MH to Creatine
ratio Prepartum
Primiparous' 1 1 1 1 1 7 1 46 1 41 1 2 0.01
Multiparous 1 01 1 42 1 22 1 45 24 0.06
Creatine
Postpartum
Primiparous 1 09 1 08 1 24 1 20 1 1 0.4
Multiparous 1 03 85 1 04 97 9 0.4
NT-MH Prepartum
Primiparous 1 92 165 220 21 5 26 0.3
Multiparous 1 73 139 1 52 162 27 0.7
NT-MH to Creatine
ratio Postpartum
Primiparous 1 70 1 53 1 74 1 76 1 1 0.3
Multiparous 1 59 1 56 145 1 65 1 2 0.8

 

 

 

‘ Contrast H vs L, and M vs MH (P < 0.01)

62

 

 

 

 

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WEI-WW

Several investigators (Marquardt et al.. 1977; Van saun et al., 1989;
Zamet at al., 1979; Johnson et al., 1981) have also shown increased DMI
in multiparous compared to primiparous cows, and this is in agreement with
the findings of this study. The increased DMI might be explained by the
fact that multiparous cows have greater b0dy size, bigger rumen capacity.
and higher requirements. Also DMI as a percentage of body weight was
different; 1 .9% for multiparous vs 1.6% for primiparous. A previous study
(Johnson et al.. 1981) also showed no effect of dry period diet on DMI
when different energy concentrations were fed. The largest decline in DMI
in this study began two weeks prepartum; which is consistent with other
studies (Bertics et al., 1992; Coppock et al.. 1972; Marquardt et al.. 1977;
Grummer et al.. 1995). This decline may be partly explained by space
occupied by uterine contents (Forbes. 1987).

Pregnant primiparous cows need more energy per Kg of BW than
multiparous cows. They use their energy for maintenance. pregnancy and
growth. where as multiparous cows use the energy only for maintenance
and pregnancy. Also. because pepartum DMI for primiparous cows was

lower than multiparous cows, therefore; calculated energy balance was

71

72

lower for primiparous cows prepartum than for multiparous cows.
However: there were no differences due to parity in the calculated energy
balance postpartum because the extra energy intake was used to produce
more milk. All groups were in negative energy balance averaged 5 wk
postpartum; due to the fact that intake was less than requirements for the
high milk yield in eariy lactation, and this was consistent with previous
studies (Lin et al., 1984).

Prepartum body fat mobilization as indicated by blood NEFA
concentrations was different for multiparous cows reflecting differences in
their energy intake. The more energy intake (HH diet) is expected to result
in a less body fat mobilization and consequently lower blood NEFA
concentration. However, it was not clear why we didn't obtain the same
results for the primiparous cows. The prepartum energy balance for
multiparous cows was higher than for primiparous cows, consistent with
their lower NEFA concentrations because blood NEFA concentrations are
inversely related to energy balance. The blood NEFA concentrations
postpartum were higher for multiparous cows than for primiparous cows,
because they mobilized more body fat to support higher milk production.
Also NEFA concentrations postpartum for all cows were higher than
prepartum; reflecting an increased demand for energy during early lactation.
Lactating cows requirements are higher than that for dry cows as indicated

by NRC (1989), so they mobilized more fat to meet the increased

73

requirements.

The increase in blood NEFA concentrations at calving time is due to
the increase in adipose tissue mobilization to meet the high energy demand
for the stressful physiological changes and forthe high milk production.

Our results are in agreement with those reported in the literature
(Johnson at al., 1981; Grummer et al., 1995)). The data indicate that
lipolysis increases at parturition to provide energy for milk production, and
that the cows continued to utilize body fat during early lactation. As in our
companion field trial (Dyke et al., 1995), the sharp increase in blood NEFA
concentration started seven days prepartum. This sharp increase happened
regardless of the fact that most of the cows were in calculated positive
energy balance. This means that there were other factors than DMI
depression which contributed to the increase in blood NEFA concentration
around calving time, as mentioned before by Vazquez-Anon at al., (1989)
and Herdt (1988) when they found that increased NEFA concentration
' started prior to DMI depression. The drop in blood NEFA concentration
postpartum reflects the end of the calving stress, recovery of DMI, and the
improvement in energy balance status of the cow.

Multiparous cows tend to mobilize more fat, as indicated by Increased
blood NEFA concentration toward calving time than primiparous cows.
Apparently this is to prepare for higher milk production. Their requirements

for Iactogenesis may be higher and their milk production peaks earlier during _

74

the lactation period.

Liver TG and fatty acids at calving were higher for multiparous cows
than for primiparous. which supports the idea that multiparous cows
mobilized more fat towards calving time to meet the higher energy
requirements for lactation. Liver TG and fatty acids were not measured
prepartum, so multiparous cows possibly started higher. Liverfatty acids
decreased with increased energy density of the diet prepartum. This means
that the more energy available from the diet results in less NEFA mobilized
from the tissue. The concentrations of T6 in the liver were smaller, and
also the percentages of fatty acids in the liver were much lower than in the
case of fatty liver. Where the T6 in fatty liver will be over 10%. which
means that those cows were in better nutritional status than the normal
cows that were analyzed by Herdt at al. (1988). They found 8% liver total

lipid concentration as percentage of wet weight. and 4% liver TG
concentration as percentage of wet weight.

The calculated energy balance prepartum was higher for multiparous -
cows than for primiparous cows , indicating that multiparous cows had
accumulated more body reserves, and hence more BCS (a measurement of
body fatness). Also multiparous cows mobilized less NEFA prepartum than
primiparous cows as discussed before. There were no observed differences
in BCS (adjusted by covarying on beginning of the experiment) due to parity

postpartum, because multiparous cows mobilized more NEFA than

75

primiparous cows.

Primiparous cows had lower body weight than multiparous cows.
because they are growing and they had not reached their mature size yet.
Body weight (8W) increased toward calving because of the increase in
conceptus growth, and may be body tissue growth. The drop in BW after
calving is inversely related to milk production, because body tissues
mobilized to produce energy for milk production. Multiparous cows lost
more weight after calving than primiparous .cows fed the same diet. may be
because they mobilized more adipose tissue for milk production after
calving, as shown by greater blood NEFA concentration and higher milk
production. I

In contrast to the report of Grummer et al. (1995), BW and BCS at
calving were not affected by dietary energy level prepartum. This may be
due to the fact that their treatment started 170 days prepartum, so the
cows had enough time to make use of the excess energy. Body condition
score data from 1 wk prepartum paralleled that of BW.

There was a positive relationship between increased dietary protein
prepartum for multiparous cows, and increased weight loss postpartum.
This may be explained by the observation that milk production peaked
earlier (6 wk) in cows fed MH and H diets compared to the cows fed L and
M diets (9 wk).

Milk yield was higher for multiparous cows, and their lactation milk

76

yield peaked earlier than primiparous cows. This is may be because they
ate more, had larger body reserves. and mobilized more adipose tissue.
which is consistent with the results of other studies (Grummer et al., 1995;
Boisclair et al., 1989: Johnson et al., 1981). The increase in diet energy
density prepartum did not affect milk yield or milk composition.

Our limited data suggests that the dry period diet with varying energy
and protein content above that recommended by NRC had no effect on
incidence of peripartum metabolic problems, and that may be due to the low
numbers of cows that we used. This is in contrast to the results of other
studies (Curtis at al., 1985) which suggested that; increasing energy and
protein content of the diet in the last 3 wk prepartum above that of NRC
requirements may reduce incidence of metabolic and reproductive disorders.
The results from the field study (Dyke et al., 1 995), which included > 2000
cows from > 90 farms, showed that; incidence of metabolic diseases was
higher with cows that had higher blood NEFA concentration around calving
time.

It is now acknowledged that the skeletal musculature is a major tissue
in whole body protein metabolism (Young at al., 1979). Creatinine is a
byproduct of muscle cell energy metabolism, and it is released from the
muscle in amounts proportional to the muscle mass. so it is used as an
indicator for muscle mass. During the breakdown of protein, 3-

methylhistidine (NMH) is released and it cannot be used for synthesis of

77

protein, so it is used as an indicator of the in vivo rate of muscle protein
breakdown in experimental animals and humans. The ratio of urinary NMH
I creatinine is used. as an estimate of myofibirillar protein degradation which
is standardized for muscle mass and for urine volume.

It was found that the rate of protein synthesis in pregnant mice is
about twice that of virgin ones, but most of the extra protein is deposited
in the fetuses and their membranes (Millican et al., 1987). The same
results were obtained in this study. where an increase in urine creatinine
concentration and a decrease in NMH I creatinine ratios were observed.
However, it is not clear why the high protein diets had higher
NMH/creatinine ratios prepartum.

The increased in NMH I creatinine ratios observed immediately after
calving can be explained as follows; body protein must be mobilized to meet
the deficiency of dietary protein postpartum. so muscle breakdown to
provide amino acids used for synthesis of milk protein 'and lactose' is
important during the first weeks of lactation. It is also possible that
increased circulating NMH in early lactation is in part the consequence of
NMH released particularly from uterine tissue during the involution process
after parturition, and also turnover increase in gastrointestinal tract in early
lactation. The was higher for primiparous cows than for multiparous cows,

may be because they are growing and more protein turnover occurred.

 

The first objective of this study was to determine If a high energy '
and/or high protein diet prepartum increases peripartum nutrient intake.
improves nutrient balance as indicated by senrm NEFA, creatinine and NMH,
and increases milk yield postpartum. It is observed in this experiment that.
dry period diet with varying energy and protein levels above NRC
recommendations had no effect on feed consumption, body condition
score, and milk production. Energy intake in primiparous and multiparous
cows increased, and prepartum plasma NEFA concentrations decreased as
nutrient density increased.

The second objective was to determine the interrelationships of plasma
NEFA and prepartum dietary energy density and protein content.

Increased dietary energy and protein for dry cows decreased plasma
NEFA two weeks prepartum. which might have decreased liver fat. Further
studies, using a larger number of cows are needed to test for the increase
of energy and to maintain the same amount of protein as in the MM diet.

Also to see the effect of these diets on peripartum diseases.

78

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