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This is to certify that the

thesis entitled

INFLUENCE OF DIETARY PROTEIN ON
PREPUBERTAL MAMMARY GLAND DEVELOPMENT
IN RAPIDLY-GROWN DAIRY HEIFERS

presented by

BRIAN KEITH WHITLOCK

has been accepted towards fulﬁllment
of the requirements for

M.S . degree in Animal Science

 

 

‘

 

mm

Méjor professor

Date 8-2-99

 

0-7639 MS U is an Afﬁrmative Action/Equal Opportunity Institution

 

LIBRARY

Michigan State
University

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE I DATE DUE DATE DUE

preterm]:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1/98 cJCIRCIDateDuopes-pu

INFLUENCE or DIETARY PROTEIN ON PREPUBERTAL
MAMMARY GLAND DEVELOPMENT IN RAPIDLY-GROWN
DAIRY HEIFERS
By

Brian Keith Whitlock

A THESIS

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

MASTER OF SCIENCE
Department of Animal Science

1999

ABSTRACT
INFLUENCE OF DIETARY PROTEIN ON PREPUBERTAL
MAMMARY GLAND DEVELOPMENT IN RAPIDLY-GROWN
DAIRY I-[EIFERS
By

Brian Keith Whitlock

My hypothesis was that high dietary protein would enhance mammary
development in prepubertal Holstein heifers fed a high-energy diet for rapid body growth
(1.2 kg/d). Heifers (n = 54) were fed a totally mixed ration of 2.85 Mcal MEI/kg with low
(14% CP; LP), standard (16% CP; SP), or high (19% CP; HP) protein from 3.5 mo of age
until slaughter at ~6 wk after puberty. Average daily BW gain for heifers on the low,
standard, and high-protein treatments were 1130, 1170, and 1180 g/d, respectively.
Dietary protein did not affect age or BW of heifers at puberty or slaughter, carcass
composition, or mammary development. Average parenchymal DNA content for heifers
on the low, standard, and high-protein treatments was 947, 1005, and 1054 mg/hemigland,
respectively. In conclusion, increasing dietary CP ﬁ'om 14 to 19% does not have a major

effect on mammary development of rapidly-grown prepubertal heifers.

ACKNOWLEDGEMENT

First, I would like to thank the many dedicated people that provided friendship,
help and guidance throughout my M.S. program. I thank my major professor, Dr. Michael
VandeHaar, for his guidance and patience as my mentor and my friend.

To the members of my guidance committee: Drs. H. Allen Tucker, Margaret
Benson and Ronald Erskine, I am grateﬁil for their contributions to my research project as
well as my graduate education as a whole.

I would like to thank Mr. Jim Liesman for his invitations to football games and his
help in the laboratory with the analysis of my data. I also thank Dr. Roy F ogwell and Mr.
Roy P. Radcliﬁ‘ for their friendship, humor, insight and ovary fondling expertise. I thank
Dr. Joe Domecq, Mr. Tom Pilbeam and Mr. Doug Mashek for assigning body condition
scores to my heifers. I thank Ms. Kristin Perkins, Mr. Luis Silva and Ms. Maria Zavala for
their ﬁiendship and assistance at the barn and the meats laboratory. To Ms. Tasha
Lapinski, Ms. Theresa Piotter, Ms. Charity Koontz and Mr. Jason Horgan, I am thankful
for their hard work and long hours at the barn and in the laboratory. I would like to thank
Mr. Lany Chapin and Dr. Chris McMahon in the Animal Reproduction Laboratory and
Mr. Dave Main and Mr. Dewey Longuski in the Forage Nutrition Laboratory for their
help and patience with various lab techniques. I thank the crews at the beef cattle research

center and the meats laboratory for their ﬁiendship and hard work.

iii

I wish to thank Michigan Soybean Promotion Committee and Michigan
Agricultural Experimental Station for the ﬁnancial support of this experiment.

I would like to express my deepest gratitude to my family. To my parents, Rex
and Mona Whitlock, I am grateﬁil for the unconditional love, strong Christian guidance,
and friendship you have always shown me. Also, thanks to my brother, Jon, and my
grandmothers, Nonie and Mammaw, for their love and support. Finally, thanks to my

wife, Lynette, for all her encouragement, love, and patience.

iv

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................ vii
LIST OF FIGURES ...................................................................................................... viii
INTRODUCTION ........................................................................................................... 1
REVIEW OF LITERATURE .......................................................................................... 3
Mammogenesis from birth to conception .............................................................. 3

Cells, tissues, and structures ..................................................................... 3

Regulation of mammogenesis .................................................................... 6

Biology of body growth ....................................................................................... 8
Effects of prepubertal diet .................................................................................. lO
Mammogenesis and milk production ....................................................... 10

MATERIALS AND METHODS ................................................................................... 15
Management of animals and treatments .............................................................. 15

Blood collection and analysis .............................................................................. 19

Tissue collection ................................................................................................ 20
Carcass composition analysis .............................................................................. 21
Mammary tissue analysis .................................................................................... 23
Statistical analysis .............................................................................................. 23
RESULTS ..................................................................................................................... 25
Live body growth and DMI ................................................................................ 25
Carcass composition ........................................................................................... 28
Mammary development ...................................................................................... 31

Serum proﬁles of somatotropin and IGF-1 .......................................................... 35

DISCUSSION ............................................................................................................... 38

Mammary development ........................................................................... 3 8
Hormones ............................................................................................... 42
External measure of mammary development ........................................... 44
Growth and DMI .................................................................................... 45
Implications ............................................................................................ 48
CONCLUSIONS ........................................................................................................... 50
APPENDIX ................................................................................................................... 51
BIBLIOGRAPHY ......................................................................................................... 53

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

LIST OF TABLES

The inﬂuence of prepubertal feeding level on mammary development in

dairy heifers ............................................................................................ 1 1
The inﬂuence of prepubertal feeding level on future milk production in

dairy heifers ............................................................................................ 11
Composition of low, standard, and high protein diets .............................. 17
Least squares means for body grth ..................................................... 26
Least squares means for carcass composition .......................................... 3O
Least squares means for mammary hemigland composition ..................... 32
Least squares means for mammary hemigland nucleic acid content .......... 33

The percentage of carcass fat, protein, and water estimated from the three-
rib cut compared with the actual values ﬁom the right halfcarcass .......... 52

vii

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

LIST OF FIGURES

Mammary development in Holstein heifers ﬁ'om birth through 12 months of
age. Modiﬁed ﬁom Sinha and Tucker (1969) ........................................... 4

Weekly body weights (top panel; 11 = 18 heifers/treatment) and dry matter
intake (bottom panel; 11 = 3 pens/treatment) of heifers fed low protein (LP),
standard protein (SP), or high protein (HP). For BW SEM = 3 and DMI,
SEM = 0.14. The arrow represents the time treatment started ................ 27

Weekly gain to feed ratio (n = 3 pens/treatment) of heifers fed low protein
(LP), standard protein (SP), or high protein (HP). SEM = 0.01. The
arrow represents the time treatments started ........................................... 29

Teat length mean for all four teats versus parenchymal mass of heifers fed
low protein (n = 15; LP), standard protein (n = 15; SP), or high protein (n
= 16; HP). 9:0.01 ................................................................................ 34

Untransformed concentrations of serum somatotropin in heifers fed low
protein (11 = 7; LP), standard protein (n = 6; SP), or high protein (n = 8;
HP) for 12 h, at 4 d before slaughter. SEM = 0.54. The arrow represents
when fresh feed was offered .................................................................... 36

Untransformed concentrations of serum IGF-I in heifers fed low protein (n
= 15; LP), stande protein (n = 15; SP), or high protein (n = 16; HP).
SEM = 15. The arrow represents the time treatments started. ............... 37

viii

INTRODUCTION

Fifteen to twenty percent of the overall expense of milk production is incurred by
heifer replacement programs (Heinrichs, 1993). Many farmers have focused on trying to
decrease the cost of rearing heifers as a means to increase farm proﬁtability. One way to
lower rearing cost is to reduce the age at ﬁrst calving (AFC). Since gestation is ~284 d,
reducing age at conception is the only way to decrease AFC. To achieve an earlier
conception age, body growth rate must increase or body size at breeding must decrease.
A smaller breeding size results in a smaller calving weight and a reduction in subsequent
milk production (Keown and Everett, 1986; and Heinrichs and Hargrove, 1987). A faster
rate of grth will enable earlier calving and optimal body size at calving.

To achieve an optimal postpartum body weight, at the recommended AFC of 24
mo, a heifer must gain ~800 g of BW/d. Body weight gains of ~1 100 g/d must be
attained during the middle of the grth curve if earlier calving (~20 mo) is to be
achieved. Whereas growing prepubertal heifers at a faster rate of gain with higher energy
diets solves the size problem, it creates another problem: namely, it decreases
mammogenesis and subsequent milk production (Swanson, 1960; Gardner et al., 1977;
Little and Kay, 1979; Sejrsen et al., 1982; Harrison et al., 1983; Petitclerc et al., 1984;

Valentine et al., 1987; Gardner et al., 1988; Peri et al., 1993; and Capuco et al., 1995).

2
However, in some studies (Capuco et al., 1995; Radcliﬁ‘ et al., 1997; and Van

Amburgh et al., 1998), growing heifers faster did not reduce mammary development or
subsequent milk production. In these studies, prepubertal dairy heifers were fed diets that
were not only high in energy but also protein.

The objective of this thesis was to determine the effect of dietary protein
concentration on body growth, carcass composition, and mammary development in

prepubertal heifers fed high-energy diets for rapid body weight gain.

REVIEW OF LITERATURE

Mammogenesis from birth to conception

Cells, tissues, and structures

In contrast to most other organs, growth and differentiation of normal mammary
glands occurs mostly in the postnatal state in a discontinous fashion. The mammary
glands of newborns contain rudimentary primary and secondary sprouts (parenchyma)
derived embryologically ﬁom ectoderrn, wheras surrounding stroma and adipose tissue
are from mesoderm. At birth the parenchyma consists of a restricted duct system,
whereas the nonglandular stroma is proportionately larger and in mature form (Tucker,
1969)

During the ﬁrst 2 to 3 months after birth, development of the heifer mammary
gland is mainly due to an increase in connective tissue and deposition of fat (Schmidt,
1971). The increase in mass parallels the increase in body growth, and this phase is
therefore referred to as isometric growth. At 2 to 3 months of age, well in advance of the
ﬁrst estrous cycle, the mammary gland begins to grow as much as three times faster than
general body growth, and this phase is referred to as allometric grth (Figure 1) (Sinha
and Tucker, 1969). Allometric mammary grth is characterized by ductal elongation

and canalization into growing stroma (Reece, 1958). Allometric growth of the mammary

Figure 1. Mammary development in Holstein heifers from birth through 12 months of
age. Modiﬁed ﬁom Sinha and Tucker (1969).

 

 

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5
gland continues for several estrous cycles after onset of puberty, at which time it returns

to an isometric growth rate until conception (Sinha and Tucker, 1969).

Prior to puberty in the rodent, expansion of the mammary ducts is the result of
rapid proliferation of the end buds. These club-shaped structures occur at the elongating
distal ends of the mammary ducts. In the mouse, end buds extend rapidly into the fat pad,
occasionally bifurcating to form new growing ducts. The end buds push between the
adipocytes until they encounter the margin of the fat pad, at which time rapid growth
ceases and the end buds regress (Williams and Daniel, 1983). After puberty, the gland
consists of an extended duct system, but alveolar development does not occur until after
conception. Alveoli are small almost spherical structures located at the end of ducts and
are made up of a single layer of epithelial cells that line the lumen of the alveoli; these are
the cells that will synthesize and secrete milk (Tucker, 1969). Although most mammary
growth occurs during pregnancy, the daughter cells that make alveolar formation possible
depend on the ducts that arise during the peripubertal period (Smith and Medina, 1988).

Mammary development is less understood in ruminants, but some distinct
differences with rodent mammary development exists. Based on Ellis and Akers (1995),
the mammary glands of prepubertal lambs lack the end buds seen in the mouse mammary
gland. The quantitative study of mammary slices of heifers (Sejrsen, et al., 1986) and
lambs (McFadden et al., 1990) demonstrate that most of the mammary fat pad of
peripubertal ruminants is devoid of epithelial ducts and the ducts in the parenchyma are
closely packed. This suggests a more complex tubular structure than in the rodent. To

use an analogy, peripubertal rodents have widely spaced mammary ducts that ﬁll the

6
mammary fat pad like the bare branches of a tree. In contrast, peripubertal ruminants

have closely packed ducts that radiate from the gland cistern in broccoli-like fashion, but
the ducts ﬁll only a fraction of the mammary fat pad. Therefore, these differences
suggest that rodent mammary development should not be assumed to accurately describe
ruminant mammary development.

Regulation of mammogenesis

Somatotropin and estrogen are essential for normal mammary development
(Lyons et al., 1958; and Cowie et al., 1966). Ovariectomy in the ﬁrst week of life
abolishes mammary growth (Wallace, 1953), and removal of the ovaries of heifers at 2.5
months of age also inhibits mammary growth (Purup et al., 1993b). This dramatic effect
of ovariectomy on mammary growth likely is due to removal of estrogen secreted by the
ovaries. Estrogen can restore growth of the mammary glands in ovariectomized heifers
(Wallace, 1953). Furthermore, Woodward and others (1993b) showed that exogenous
estradiol stimulates proliferation of mammary epithelial cells in gonadally intact
prepubertal heifers. However, diﬂ‘erences between circulating levels of estradiol in intact
(0.41 pg/ml) and ovariectomized (0.31 pg/ml) prepubertal heifers are small (Purup et al.,
1993b). This difference in estradiol concentration seems too small to be responsible for
the complete block of mammary growth in ovariectomized heifers. In addition,
mammary explant cultures are more likely to proliferate in response to estradiol than
isolated mammary epithelial cells (Purup et al., 1993b). Woodward and others (1993a)
observed that treatment of bovine ﬁbroblasts with estradiol induced secretion of a

substance that stimulates proliferation of mammary cells. The idea that estrogen’s

7
stimulation of epithelial cell proliferation is mediated by autocrine and/or paracrine

secretion of growth factors is referred to as the estromedin hypothesis (Sirbasku, 1978).
Asynchronous secretion of estrogen and progesterone is a possible cause of the return of
mammary growth to an isometric rate after puberty (Tucker, 1981). However,
immunization of heifers against gonadotropin-releasing hormone, which decreases serum
estrogen and progesterone, does not alter mammary growth (Sejrsen, 1994).

The anterior pituitary hormones are absolutely critical for mammary development.
Utilizing ablation/replacement experiments, Talwalker and Meites (1961) reported that
hypophysectomy reduces mammary development in rats and exogenous prolactin and
somatotropin restores mammary development. In addition, exogenous somatotropin
increases mammary growth in ruminants (Sejrsen, 1986; Sandles and Peel, 1987;
McFadden et al., 1990; Glasser et al., 1991; and Radcliff et al., 1997), but there is little
evidence that somatotropin has a direct effect on the mammary gland. Although
somatotropin receptor mRN A as been detected in bovine mammary tissue (Glimm et al.,
1990), research has failed to detect somatotropin receptors in ruminant mammary tissue
(Keys and Djianne, 1988; McFadden et al., 1990; and Purup et al., 1995). In addition,
somatotropin does not stimulate proliferation of isolated bovine epithelial cells (Collier et
al., 1993). Most evidence suggests that somatotropin does nOt act directly but rather it
acts indirectly on the mammary gland by factors such as insulin-like growth factor-I
(IGF-I) (Akers, 1990).

Administration of somatotropin in cattle increases serum IGF-I concentrations

(Bauman and Vernon, 1993; Dahl et al., 1993; Pump et al., 1993a; Sharma et al., 1994;

8
Vanderkooi et al., 1995; Sharma et al., 1996; and Yung et al., 1996), and liver IGF-I

mRNA abundance (Sharma et al., 1994; Vanderkooi et al., 1995; and Sharma et al., 1996)
but has no effect on IGF-I mRN A abundance in the mammary gland (Sharma et al.,
1994). Receptors for IGF-I are present in mammary tissue (Purup et al., 1995) and in
isolated bovine mammary cells (Romagnolo et al., 1994), and IGF-I stimulates
proliferation of isolated bovine mammary epithelial cells (Zhao et al., 1992; Romagnolo
et al., 1992; and Collier et al., 1993). In addition, there are at least six different IGF-I
binding proteins (IGFBPs) that modulate the biological activity of IGF-I (Clemmons,
1997). Sharma et al. (1997) reported that administration of somatotropin to prepubertal
heifers decreased hepatic mRN A and serum concentrations of IGFBP-2 ~70 and ~54%,
respectively. In addition, these changes were associated with increases in prepubertal
mammary development (Radcliff et al., 1997). Thus, local production of IGF-I and/or
IGFBPs in the mammary gland may modify the relationship between somatotropin, IGF-I

and mammary growth.

Biology of body growth

Growth is the increase in the physical size of the body and increase in the total
weight of the muscle and various internal organs. Growth results ﬁ'om an increase in
both size and number of cells. An increase in number of cells is called hyperplasia; an
increase in cell size is called hypertrophy. The various body parts grow in an orderly
manner, with the various tissues growing at different rates from birth to maturity. One

organ or tissue may begin rapid growth at a time when the growth rate of other organs or

9
tissues is slower. For example, the maximum rate of growth of the nervous system

occurs early in development, and is followed by the skeleton, then by muscle, and ﬁnally
by adipose tissue.

Shortly after birth, bone grows more rapidly than other tissues in proportion to
total body weight. This rapid rate of skeletal growth decreases dramatically by 8 to 10
months of age in cattle (Berg and Butterﬁeld, 1968). However, new bone is constantly
being laid down and reabsorbed by the body throughout life. An equilibrium between
new growth and reabsorption maintains mature bone size.

Maximum muscular development occurs later than maximum skeletal growth.
Growth of muscle as a function of time is a sigmoidal curve: i.e., at ﬁrst growth is slow,
then increases dramatically before slowing again. Postnatal muscle growth is a process
of hypertrophy and not hyperplasia (Burleigh, 1974; and Goldspink, 1974). As a
myoﬁber grows, nuclei are added ﬁ'om mitosis of satellite cells, which reside between the
basement membrane and the sarcolemma. Proteins are continually being synthesized and
degraded and the balance between these two processes dictates the rate of protein
deposition and muscle growth. When protein synthesis is greater than protein
degradation, protein accretion or net muscle growth occurs.

Accretion of adipose tissue increases as the rate of muscle grth declines. The
major function of lipid in an animal is long-term storage of energy (Leat and Cox, 1980).
Thus, as net growth of bone and muscle stops, adipose accretion may continue as long as

nutrient availability permits (Hammond, 1960). Similar to muscle, accretion of adipose

10
tissue is dependent upon rates at which lipid is deposited (lipogenesis) and removed

(lipolysis).

Effects of prepubertal diet

Mammogenesis and milk production

Previous studies indicate that rapid body growth in prepubertal heifers impairs
mammary development (Table l) (Sejrsen et al., 1982; Harrison et al., 1983; Petitclerc et
al., 1984; Valentine et al., 1987; and Capuco et al., 1995) and subsequent milk production
(Table 2) (Gardner et al., 1977; Little and Kay, 1979; Valentine et al., 1987; Peri et al.,
1993; and Van Amburgh et al., 1998). This decrease in mammogenesis and milk
production associated with accelerated rates of gain is often attributed to truncated
parenchymal development and excess fat deposition in the mammary gland (Swanson,
1960). Prepubertal growth rate is inversely related to age at puberty (Schillo et al., 1992).
Therefore, rapidly-grown prepubertal heifers will attain puberty before conventionally-
reared heifers. Since mammary growth returns to an isometric rate shortly after puberty
(Sinha and Tucker, 1969), reducing age at puberty by rapid prepubertal growth will
truncate the allometric growth phase and thus may impair mammary development. For
example, Sejrsen et al. (1982) fed pre- and postpubertal heifers a 15% CP diet for ad
libitum or restricted intake to produce 1200 and 600 g of BW gain/d, respectively. All
prepubertal heifers were slaughtered at 320 kg BW and heifers fed for ad libitum intake
had ~3 0% less mammary parenchymal DNA compared with heifers on restricted feeding.

However, treatment did not effect mammary development of postpubertal

Table l. The inﬂuence of prepubertal feeding level on mammary development in dairy
heifers.

 

BW gain
Study ng CPrMEl % of control2
Sejrsen et al., 1982 1270 52 68%
Harrison et al., 1983 1180 43 59%
Petitclerc et al., 1984 1030 48 75%
Valentine et al., 1987 950 52 59%
Stelwagen and Grieve, 1990 1000 58 115%
Capuco et al., 1995 1010 54 52%
Capuco et al., 1995 970 83 94%
Radcliff et al., 1997 1190 68 102%

 

I g crude protein/Mcal metabolizable energy.
2 Mammary development of rapid gain group relative to control group.

 

Table 2. The inﬂuence of prepubertal feeding level on future milk production in dairy
heifers.

 

BW gains
Study ng CP:I\/II-3l % of control2
Gardner et al., 1977 1100 55 82%
Little and Kay, 1979 1090 43 48%
Valentine et al., 1987 950 52 72%
Peri et al., 1993 1090 51 84%
Radcliff et al., 1998 1200 68 88%
Van Amburgh et al., 1998 950 64 95%

 

I g crude protein/Meal metabolizable energy fed during prepubertal treatment period.
2 First lactation milk production of cows grown rapidly during the prepubertal period
relative to cows grown at conventional rates during the prepubertal period.

12
heifers. Thus, the critical period in which excessive rate of gain reduces mammary

development and ﬁxture milk production is before puberty.

As Table 1 shows, responses of mammary growth to rapid prepubertal BW gains
in the literature have been inconsistent and ranges from no impairment to 60% less
mammary tissue than conventionally grown heifers. Some of the variation might be
explained by differences in laboratory techniques used to quantify mammary
development. For example, Stelwagen and Grieve (1990) measured total mammary
DNA instead of DNA in parenchyma. The different responses might also be the result of
differences in length of treatment periods or the dietary methods implemented to achieve
rapid gains. For example, some reports indicate that the ratio of protein to energy in the
diet may explain some of the variation in effects of accelerated prepubertal growth on
mammary development and subsequent milk production (Capuco et al., 1995; Pirlo et al.,
1997; Radcliff et al., 1997; and VandeHaar, 1997).

Capuco et al. (1995) fed prepubertal Holstein heifers either an alfalfa-based high-
protein diet (83 g CP/Mcal ME) or corn silage-based low—protein diet (54 g CP/Mcal
ME) to achieve rapid (~1000 g/d) or control (~700 g/d) BW gains. Heifers were killed
when they weighed at least 325 kg and had completed two or more estrous cycles.
Mammary development was impaired 48% when accelerated) gains were produced in
heifers fed the corn silage-based diet but no impairment of mammary development when
accelerated gains were produced by the alfalfa-based diet. Radcliff et al. (1997) fed
prepubertal Holstein heifers a high-energy, high-protein (68 g CP/Mcal ME) diet to

produce ~1200 g of BW gain/d or a control diet to produce ~800 g of BW gain/d ﬁ'om 4

1 3
mo of age until the ﬁfth estrous cycle. Compared with control heifers, heifers fed the

high-energy, high-protein diet reached puberty and were killed 1.6 mo earlier. However,
the accelerated prepubertal grth rate did not impair mammary development. In a study
by Pirlo et al. (1997), heifers were fed diets that were 10% above or below NRC (1989)
recommendations for protein and energy ﬁ‘om 100 to 300 kg BW. Heifers fed the high
energy with high protein produced 5% more fat-corrected milk than heifers fed the high
energy with low protein, and they produced 95% as much fat-corrected milk as control
heifers. VandeHaar (1997) examined the relationship between mammary development or
milk yield and the dietary protein to energy ratio from 11 studies in which rapid gain
heifers exceeded 900 g BW gain/d. Estimated dietary protein to energy ratios varied
considerably among the studies, from 43 to 83 g CP/Mcal ME. Across studies, mammary
development of rapidly-grown heifers relative to their controls was positively correlated
with the protein to energy ratio of the diets they were fed. Furthermore, the protein to
energy ratio accounted for 51% of the variation in mammary parenchyma responses and
78% of the variation in milk yield responses to rapid growth rate.

A major mediator of dietary effects on mammary development may be differences
in endogenous hormone secretions. For example, Sejrsen et al. (1983) reported that
serum somatotropin concentrations and mammary development were reduced in
prepubertal heifers fed for ad libitum intake compared with heifers fed restricted intake.
Sejrsen hypothesized that the decreased somatotropin concentrations were responsible for
the impaired mammary parenchymal growth observed for heifers fed a high-energy diet

for ad libitum intake. However, when Capuco et al. (1995) gave prepubertal heifers free

l4
access to an alfalfa-based diet to produce rapid BW gains, serum somatotropin

concentrations only tended to be decreased and mammary development was not impaired.
Whereas, accelerated growth ﬁom ad libitum intake of a corn silage-based diet decreased
serum somatotropin concentrations and mammary development 25 and 48%,
respectively. Similarly, when Radcliff et al. (1995) fed prepubertal heifers a high-protein
diet to produce ~1200 g of BW gain/d, serum somatotropin concentrations and mammary
development were unaffected compared with control heifers. These studies suggest that
inadequate protein might have been responsible for the impaired mammary development
and subsequent milk production of heifers grown rapidly before puberty in previous

literature.

15

MATERIALS AND METHODS

Management of animals and treatments

Sixty-four Holstein heifers [approximate age = 11 wk and mean BW (iSEM) =
101 i 1 kg] were purchased in 3 consecutive weeks between May 4 and May 19 (~20
heifers/wk): each week being a different age block. All animals were allowed 30 d to
acclimate to new surroundings and were monitored for illnesses at the Michigan State
University Dairy Teaching and Research Center. Heifers were grouped by age block and
allowed free access to an outside paddock during the acclimation period. On the ﬁrst
acclimation day all heifers were injected with 10 mg/kg BW of Micotilm (Elanco Animal
Health, A Division of Eli Lilly and Company) as a prophylactic. Rectal temperatures
were determined for the ﬁrst 5 d of the acclimation period. Heifers were injected with
Micotil“D a second time if rectal temperatures were above 397°C. During the ﬁrst half of
the acclimation period heifers were fed the same 15% CP complete feed and alfalfa-
orchard grass hay they received prior to purchase. During the second half of the
acclimation period heifers were fed an adjustment ration that initially contained 75% of
the previously fed complete feed and 25% of a total mixed ration similar to the treatment

diets. The percentage of complete feed was decreased over 1 wk until the diet was solely

16
the total mixed ration. During the last wk of acclimation heifers were fed the total mixed

ration.

Following the acclimation period, heifers were transported to the Michigan State
University Beef Cattle Research Center. Within each age block, the eighteen heifers with
the greatest rate of BW gain during the acclimation period were blocked by BW into
groups of three and randomly assigned to one of three treatments. All heifers for a given
treatment within each age block were housed in the same pen. Thus, three pens of six
heifers (one pen per age block) represented each of the three treatments. Treatments
began at 106 d of age and continued until the early luteal phase of the fourth estrous
cycle.

Each of the three treatment diets was 40% alfalfa-grass haylage and 60% grain
and'contained ~2.85 Mcal ME/kg. This energy density was expected to produce 1200 g
of BW gain/d when diets were fed ad libitum. Diets were low (14% CP; n = 18; LP),
standard (16% CP; n = 18; SP), or high (19% CP; n = 18; HP) protein. The alfalfa-grass
haylage was the ﬁrst-cutting ﬁom a single ﬁeld and harvested and stored in a bag during
the early bloom period. Haylage samples were collected twice a week to assess dry
matter content and haylage was collected every other week to assess protein and ﬁber
content. Samples of ground corn, soybean meal-48, and expeller soybean meal were
collected upon purchase to assess protein and energy content. Composition of diets based
on actual analysis is described in Table 3. Diets were fed as a TMR ﬁesh every day
between 0900 and 0930 h and heifers had free access to water and the respective diet.

Orts for each pen were collected at 0700 h and weighed daily. Mean dry matter intake

l 7
Table 3. Composition of low, standard, and high protein diets.

 

Low Standard High
Ingredients, % of DM
Alfalfa-grass haylage] 40.0 40.0 40.0
Ground cm2 54.0 48.1 42.2
Soybean meal3 5.0 5.0 5.0
Expeller soybean meal4 0.0 5.9 11.8
Minerals and vitamins5 1.0 1.0 1.0
Nutrient composition
NDI‘, % ofDM 25.1 25.4 25.6
ADI-7, % ofDM 13.9 14.3 14.7
ME“, Meal/kg 2.85 2.85 2.85
NEm”, Meal/kg 1.90 1.91 1.91
NEglo, Mcal/kg 1.26 1.27 1.27
CP, % ofDM 13.7 16.2 18.8
RUP“, % ofCP 33.4 36.0 37.9
AP”, % ofDM 10.6 11.6 12.7
CPzME, (g CP/Mcal ME) 48.0 57.0 66.0

 

1 Haylage was analyzed every 2 wk and contained 15.3% CP (i 0.8 SD), 47.1% NDF (i
1.6 SD) and 30.0% ADF (:l: 0.8 SD) and energy values were estimated to be 2.43 Mcal
ME/kg, 1.55 Mcal NEm/kg, and 0.95 Mcal NEg/kg.

2 Ground corn was analyzed twice and contained 9.5% CP (3: 0.5 SD), 11.4% NDF (i
0.1), and 3.0% ADF (i 0.0) and energy values were estimated to be 3.18 Mcal ME/kg,
2.17 Mcal NEm/kg, 1.50 Mcal NEg/kg.

3 Soybean meal-48 was analyzed once and contained 53.2% CP, 6.9% NDF, and 5.2%
ADF and energy values were estimated to be 3.26 Meal ME/kg, 2.24 Mcal NEm/kg, 1.55
Mcal NEg/kg.

1 8
4 Expeller soybean meal was all from the same batch and was analyzed once. It

contained 53% CP, 8.0% NDF, and 4.2% ADF and energy values were estimated to be
3.21 Mcal ME/kg, 2.20 Mcal NEm/kg, 1.52 Mcal NEg/kg.

5 Mineral and vitamin mix contained 23.8% white salt, 10.4% trace mineral-vitamin
premix and 4.0% DECCOX-lO Etts (Purina Mills; .5% decoquinate) and was formulated
so the diet provided 100% mineral and vitamin requirement (NRC 1989).

6 Neutral detergent ﬁber.

7 Acid detergent ﬁber.

8 Metabolizable energy.

9 Net energy for maintenance.

1° Net energy for gain.

” Rumen-undegraded protein using book values of 21%, 50%, 30%, and 50% of CP for
alfalfa-grass haylage, ground corn, soybean meal, and expeller soybean meal.

‘2 Absorbed protein = .8[.8(MCP g/d) + (%RUP as %CP)(CP g/d)], MCP = (38
g/Mcal)(ME intake Meal/d).

for a pen was recorded. Heifers were exposed to ambient temperatures and photoperiod
from the time of purchase until slaughter.

All heifers were weighed at ~0800 h before feeding on 2 consecutive (1 each week
to monitor BW gain. The mean of the two weights was then assigned as a heifer’s
weekly weight. Weekly weights were used to calculate average daily BW gains. The
height at the withers was measured every 2 wk. Body condition score (BC S) was
assessed using a ﬁve-point scale (1 = thin, 5 = fat; Wildman et al., 1982) every 4 wk by
three experienced examiners. The three scores for each heifer were averaged and

assigned to that heifer as her monthly score.

19
Commencing when a heifer attained 215 kg of BW or 7 mo of age, which ever

was ﬁrst, her reproductive status was examined weekly to determine onset of puberty. A
heifer was considered pubertal when a corpus luteum (CL) was detected on either ovary
by palpation through the rectum. If a CL was present again 21 (1 following the ﬁrst CL,
heifers were injected with 25 mg of PGan (Lutalyseg, Pharmacia & Upjohn Inc.) 3 d
later. Eleven (1 after their ﬁrst injection of PGan, heifers were rectally palpated again.
If a CL was present they received another injection of PGan. Eleven (1 after the second
injection of PGan, heifers were rectally palpated again. If a CL was present,
heifers were killed on the following day or d 47 after detection of the ﬁrst CL.

On the day of slaughter, heifers were transported to the Michigan State University
Meats Laboratory at ~063O h. All heifers were weighed, stunned by captive bolt, and
killed by exsanguination between 0700 and 1000 h. The number of heifers killed each
week depended on the date for detection of the ﬁrst corpus luteum and ranged from 1 to 6

heifers. All heifers were slaughtered at a mean of 46 i 0.7 d after detection of the ﬁrst

corpus luteum.

Blood collection and analysis

Blood samples (~10 ml) were collected every 4 wk at ~0800 h via jugular
venipuncture with VacutainersG (Becton Dickinson & Co., Rutherford, NJ). All samples
were stored at room temperature (~21°C) for ~6 h and then at 4°C ~15 h. Serum was
harvested after centrifugation at 1550 x g and 4°C for 25 min and then frozen at -20°C.

Serum samples were assayed for insulin-like growth factor-I concentrations using

20
ethanol/acid extraction (Bruce et al., 1991) and radioimmunoassay according to GroPep

Pty Ltd (Growth Factors Products and Protocols, Adelaide, Australia) with GroPep’s
IGF-I standard and primary antibody but modiﬁed as in Sharma et al. (1994) with
Staphylococcus aureus used in place of the secondary antibody.

For slaughter dates in which each treatment was represented by at least one heifer,
intensive sampling of blood for assessment of the proﬁle of somatotropin concentration
was completed 4 d before slaughter. Heifers were ﬁtted with sterile indwelling jugular
catheters (18 gauge; loo-Rally, Palo Alto, CA) 5 d before slaughter. On the following d,
serial blood samples were collected at 20-min intervals for 12 h (0700 to 1900 h).
Twenty-one heifers, seven from the low-protein diet, six ﬁ'om the standard-protein diet,
and eight from the high-protein diet, were bled for assessment of the proﬁle of
somatotropin concentration. Catheter patency was maintained between samples by
ﬂushing the catheter with 3.5% sodium citrate in sterile water. Serum was harvested and
samples were stored as previously mentioned until serum concentrations of somatotropin

were quantiﬁed using radioimmunoassay as in Gaynor et al. (1995).

Tissue collection

Mammary glands were quickly removed from the carcass and placed on a table
with the ventral surface up. A metric ruler was used to measure from the base of each
teat to its tip. The glands were bisected along the median suspensory ligament into right

and left halves. The left half was weighed, placed in a plastic bag, and frozen by

21
submersion in a tub of dry ice and 95% ethanol. Frozen hemiglands were stored at -20°C

until analyzed.

The digestive tract was removed ﬁ'om the carcass, the gallbladder was removed
ﬁ'om the liver, and the liver was weighed. The intestines were separated ﬁ'om the upper
gastrointestinal tract at the pylorus. Approximately 80 to 90% of the fat was removed
ﬁom the upper gastrointestinal tract and saved. Digesta were ﬂushed ﬁ'om the intestines
with water, and the intestines were cut several times to remove water. The intestines with
omental fat were combined with the fat from the upper gastrointestinal tract and stored at
-20°C until grinding and analysis of lipid content.

After the hide was removed, the carcass was split into halves along the vertebral
column. The carcass halves were then weighed. Perirenal fat was removed from the left
half beginning at the 4th lumbar vertebra and proceeding forward to the adrenal gland

and then weighed. The carcass was washed and stored at 2°C.

Carcass composition and analysis

Twenty-four h after slaughter, pelvic area was calculated from two linear
measurements of the left half of the carcass, one from the ventral edge of the third
coccygeal vertebrae to the symphysis pubis and a second at 90° from the midsagittal
plane of the carcass to the middle of the pelvic wall. The second measurement was
multiplied by two to represent the total width of the pelvic opening and then multiplied

by the ﬁrst measurement to estimate total pelvic area (Radcliff et al., 1997).

22
The left half of each carcass was cut between the 7th and 8th ribs and between the

12th and 13th ribs. The rib section, including ribs 8 through 12, was removed. The
section containing ribs 9, 10, and 11 was then dissected (Hankins and Howe, 1946),
weighed, and deboned. In addition to the rib sectiOns ﬁom each carcass, the entire right
half of 12 animals was deboned. Bone and soft tissue were weighed. Soft tissue was
ground, mixed and subsampled for analyses of protein, fat, and water content. Crude
protein content was determined in fresh samples by combustion and subsequent
measurement of thermal conductivity using the Combustion method (AOAC, 1990,
Method 990.03) utilizing the combustion nitrogen analyzer LECOG FP-ZOOO (Leco
Corporation, St. Joseph, MI). Fat was determined by Soxhlet ether extraction of ﬂesh
samples (AOAC, 1990). Water was determined by the difference in weight after drying
fresh samples in an oven at 110°C for 24 h. Carcass protein, fat, and water contents were
estimated using equations based on the ninth-tenth-eleventh-rib cut (Hankins and Howe,
1946). Equations, were Y = 5.64 + .69X, Y = 2.73 + .78X, and Y = 14.28 + .78X, for the
protein, fat, and water, respectively, where Y was the edible portion of the dressed heifer
carcass, and X was the edible portion of the heifer three-rib cut. These estimates were
compared with the actual measurements of the right half of 12 animals.

Ruminal fat and intestines were weighed and ground at the Michigan State
University Poultry/Mink Farm. The ground tissue was mixed and subsampled for
analyses of fat content. Fat was determined by Soxhlet ether extraction of ﬂesh samples

(AOAC, 1990).

23
Mammary tissue analysis

The frozen left half of the udder was cut transversely with a band saw into 5— to
IO-mm thick slices. All slices ﬁom both the anterior and posterior ends of the gland that
did not contain parenchymal tissue were discarded. Slices were allowed to thaw slightly,
and skin, teats, and supramammary lymph nodes were dissected. Fat located beyond the
border of the parenchyma (in those slices that contained parenchyma) was removed and
weighed. This fat was deﬁned as extra parenchymal fat. The remaining tissue was
referred to as parenchymal tissue. Frozen parenchymal tissue was weighed and ground
with liquid nitrogen into a ﬁne powder with a blender. The powder was mixed and
subsampled for subsequent analysis of DNA and RNA content (Tucker, 1964), dry
matter, protein, and fat. Dry matter was determined by the difference in weight after
drying fresh samples in an oven at 110°C for 24 h. Crude protein content was determined
in fresh samples by combustion and subsequent measurement of thermal conductivity
using the Combustion method (AOAC, 1990, Method 990.03) utilizing the combustion
nitrogen analyzer LECOQ FP-ZOOO (Leco Corporation, St. Joseph, MI). Fat was

determined by Soxhlet ether extraction of ﬂesh samples (AOAC, 1990).

Statistical analysis
Data for mean live body growth from d 0 until slaughter, carcass composition,
mammary composition, and mammary nucleic acid content were analyzed by the GLM

procedure of SASQ (1996). The model included treatment as a ﬁxed effect and group as a

random effect using the following equation:

24
Y = treatment + group + group*treatment + residual.

treatment = low, standard, and high protein diet

group = A, B, and C age group

residual = animal(group*treatment)

Differences were determined using orthogonal contrast for the linear and quadratic
effects of the diet using the CONTRAST statement in PROC GLM of SAs° (1996).
Least squares means and standard errors of least squares means are presented.

Mean concentrations of somatotropin and IGF-1 were transformed by natural
logarithm to eliminate heterogenous variance. Natural logarithm transformed
concentrations of somatotropin and IGF-1 were analyzed by the GLM procedure of SASG
(1996) using the following equation:

Y = treatment + group + group*treatment + animal(group*treatment) + time +

time*treatment + residual.

treatment = low, standard, and high protein diet

group = A, B, and C age group

residual = time* group + time*group*treatment + animal(time*group*treatment)
Values presented for concentrations of somatotropin and IGF-1 are means of

untransformed data.

25

RESULTS

Eight heifers, three from both the low and standard protein diets and two from the
high protein diet, were removed from the experiment. Four heifers were removed
because they were freemartins’; one from each of the low and high protein diets and two
were ﬁom the standard protein diet. Two heifers were removed due to complications
ﬁom rectal palpation; both were from the low protein diet. Two heifers were removed
ﬁ'om the experiment because of late onset of puberty; one was from both the standard and
high protein diets. Treatments continued for 165, 164, and 160 i 8 d for heifers receiving

the low, standard and high protein diet, respectively (Table 4).

Live body growth and DMI

Initial measures of age, BW, height at the withers, and BCS were not different
among treatment groups (Table 4). Figure 2 illustrates that dry matter intake and BW
were unaffected by increasing dietary protein. Diet did not inﬂuence body weight at
slaughter or ﬁnal height at the withers. Since age at slaughter was similar for treatment
groups, rates of BW and withers height gain were unaffected by the differences in dietary
protein (Table 4). Because changes in dietary protein to energy did not inﬂuence DMI

or BW gain, no difference in feed efﬁciency resulted with the gainzfeed ratio for the low,

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27

Figure 2. Weekly body weights (top panel; 11 = 18 heifers/treatment) and dry matter
intakes (bottom panel; 11 = 3 pens/treatment) of heifers fed low protein (LP), standard
protein (SP), or high protein (HP). For BW, SEM = 3 and for DMI, SEM = 0.14. The
arrow represents the time treatment started.

300 "

250 ‘

Body weight (kg)
N
6
O

150 "

 

 

100 "

DMI (kg/heifer/d)
at

 

 

 

15 17 19 21 23 25 27 29 31 33
Age (wk)

28
standard and high protein diet being 0.19, 0.20, and 0.20 kg BW gain/kg DMI,

respectively (Figure 3). Similarly, change in body condition score throughout the
experiment was unaffected by treatment. Dietary protein also did not affect age or body
weight at the time of observed ﬁrst corpus luteum. In contrast, diet altered pelvic area at

slaughter. Heifers fed the standard-protein diet had greater pelvic areas than the other

groups.

Carcass composition

Carcass weights and carcass weights as a percentage of live BW were similar
among treatment groups (Table 5). Diet did not inﬂuence total liver weight or liver
weight as a percentage of BW. As assessed using equations based on the edible portion
of the ninth-tenth-eleventh-rib cut, percentage of carcass protein and total protein in the
carcass were not affected by treatment. The standard diet decreased percentage of
carcass fat relative to the low and high protein diets. Diet did not affect total perirenal or
omental-intestinal fat.

In addition, diet did not inﬂuence the percentage of carcass protein, fat, or water
as assessed by actual carcass analysis of 12 animals. Using equations based on the edible
portion of the ninth-tenth-eleventh-rib cut (Hankins and Howe, 1946), the estimated
mean percentage of carcass protein, fat, and water of 12 heifers was 17.9 i 0.3, 20.0 i
0.9, and 60.3 i 0.7, respectively. The mean percentage of carcass protein, fat, and water
determined from the edible portion of the right half carcass of the same 12 heifers was

18.9 i 0.3, 17.9 i 1.1, and 63.8 i 1.0, respectively. The correlation coefficients between

29

Figure 3. Weekly gain to feed ratio (11 = 3 pens/treatment) of heifers fed low protein
(LP), standard protein (SP), or high protein (HP). SEM = 0.01. The arrow represents the
time treatments started.

0.35 -

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Gain : Feed
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31
the percentage of carcass protein, fat, and water estimated from the edible portion of the

ninth-tenth-eleventh-rib cut (Hankins and Howe, 1946) and from the edible portion of the
right half carcass were: r = 0.29 for percentage of carcass protein, 0.71 for fat and 0.46

for water.

Mammary development

The mass of dissected parenchyma and parenchyma as a percentage of BW was
~10% greater for heifers fed the high protein diet than those fed low protein; however,
this difference was not statistically signiﬁcant. The total mass of extra-parenchymal fat
as well as extra-parenchymal fat as a percentage of BW was similar among treatment
groups. In addition, diet did not affect lipid, dry-fat-free tissue, or protein in the
parenchymal tissue (Table 6).

Similar to the total mass of dissected parenchyma, the parenchymal DNA and
RNA content was ~10% greater, but not significantly, in heifers fed the high protein diet
relative to heifers fed the low protein diet (Table 7). Similarly, parenchymal DNA and
RNA content as a percentage of BW was greater for heifers fed the high protein diet
compared with heifers fed the low protein diet, but this was not statistically signiﬁcant.
Diet did not affect concentrations of DNA or RNA in parenchymal tissue, and the

RNA/DNA ratio was similar among treatment groups.

 

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34
In addition to measuring dissected parenchyma mass, mammary cell numbers and

metabolic activity, teat length was measured as a possible gauge of mammary
development. Diet did not inﬂuence front or rear teat length (Table 6), and variation in

teat length accounted for only 1% of the variation in mammary parenchymal mass

(Figure 4).

Figure 4. Teat length mean for all four teats versus parenchymal mass of heifers fed low
protein (n = 15; LP), standard protein (n = 15; SP), or high protein (11 = 16; HP). 13 =
0.01.

 

 

 

60 "
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50 " A
Am
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100 200 300 400 500 600 700

Parenchymal Mass (g / hemigland)

35
Serum profiles of somatotropin and IGF-1

Proﬁles of somatotropin concentration in serum for 12 h, at 4 d before slaughter
are presented in Figure 5. Increasing the ratio of dietary protein to energy did not affect
average somatotropin concentrations during the day or the feeding induced decrease in
somatotropin conentrations. Proﬁles of IGF-I concentrations in serum are presented in

Figure 6. Concentrations of IGF-I were not different among treatment groups.

36

Figure 5. Untransformed concentrations of serum somatotropin in heifers fed low
protein (n = 7; LP), standard protein (n = 6; SP), or high protein (n = 8; HP) for 12 h, at 4
d before slaughter. SEM = 0.54. The arrow represents when fresh feed was offered.

 

 

10 "
-9—LP
‘B—SP

8 ’ +11]:

2 o

5

56 '

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a.

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700 900 1100 1300 1500 1700 1900
Time of day

37

Figure 6. Untransformed concentrations of serum IGF-I in heifers fed low protein (n =
15; LP), standard protein (11 = 15; SP), or high protein (n = 16; HP). SEM = 15. The
arrow represents the time treatments started.

500 -
45° ' —e—LP
400 P -E-SP

 

 

 

 

100 I I I p I I I

80 ' 110 140 170 -90 -60 -30 0
Chronological age ((1) Time relative to kill ((1)

38

DISCUSSION

Mammary development

The present study is the ﬁrst to directly show that dietary protein does not have a
signiﬁcant affect on mammary growth in rapidly growing prepubertal dairy heifers.
Level of dietary protein did not alter mammary grth regardless of how it was
measured: parenchymal mass, protein, dry-fat-free tissue, DNA, or RNA.

Several experiments (Gardener et al., 1977; Little and Kay, 1979; Sejrsen et al.,
1982; Harrison et al., 1983; Petitclerc et al., 1984; Valentine et al., 1987; Gardner et al.,
1988; and Peri et al., 1993) demonstrated that rearing prepubertal dairy heifers at
accelerated grth rates reduced mammary development and subsequent milk
production. However, other studies, in which high protein diets were fed, found that
rearing dairy heifers at accelerated rates of BW gain did not impair mammogenesis
(Capuco et al., 1995; and Radcliff et al., 1997) or subsequent lactation (Van Amburgh et
al., 1998). Sejrsen et al. (1982) fed prepubertal heifers a 15% CP (52 g CP/Mcal ME)
diet at ad libitum or restricted intake to produce 1200 and 600 g of BW gain/d,
respectively. Heifers were slaughtered at 320 kg BW and those fed ad libitum had ~3 0%
less mammary parenchymal DNA than those on restricted feeding.

Capuco et al. (1995) fed prepubertal Holstein heifers either an alfalfa-based high

protein diet (83 g CP/Mcal MB) or corn silage-based low protein diet (54 g CP/Mcal ME)

39
to achieve rapid (~1000 g/d) or control (~700 g/d) BW gains. Heifers were killed when

they weighed at least 325 kg and had two or more estrous cycles. Mammary
development was impaired 48% when accelerated gains were produced in heifers fed the
corn silage-based diet but no impairment of mammary development occurred when
accelerated gains were produced by the alfalfa-based diet. Although the authors
speculated that the different responses were caused by the difference in bulk densities of
the corn silage-based and alfalfa-based diets, the low protein of the corn silage diet seems
a more likely explanation. In addition, Radcliff et al. (1997) fed prepubertal Holstein
heifers a high-energy, high-protein (68 g CP/Mcal ME) diet to produce ~1200 g of BW
gain/d or a control diet to produce ~800 g of BW gain/d from 4 mo of age until the ﬁlth
estrous cycle after puberty. Heifers fed the high-energy, high-protein diet had mammary
development similar to that of control heifers.

VandeHaar (1997) examined the relationship between mammary development or
milk yield and the dietary protein to energy ratio from 11 studies in which gains of
heifers exceeded 900 g of BW/d. Estimated dietary protein to energy ratio varied
considerably among the studies, ﬁ'om 43 to 83 g CP/Mcal ME. Across the studies,
mammary development of rapidly-grown heifers relative to their controls was positively
correlated with the protein to energy ratio of the diets. Furthermore, the protein to energy
ratio accounted for 51% of the variation in mammary parenchyma responses and 78% of
the variation in milk yield responses to rapid growth rate. These reports suggest that
inadequate protein might be responsible for the impaired mammary development and

subsequent milk production of rapidly-grown prepubertal heifers. However, this is the

40
ﬁrst study to directly investigate the effect of dietary protein on mammary development

in heifers fed isocaloric diets to achieve rapid rates of BW gain.

Based on previous literature (Table 1), I expected mammary development to be
impaired 30 to 50% in rapidly-grown prepubertal heifers fed 14% CP (48 g CP/Mcal ME)
compared with 19% CP (66 g CP/Mcal ME). Although parenchymal DNA was 10%
greater in heifers fed 19% CP compared with heifers fed 14% CP, this difference was not
close to statistical signiﬁcance. Thus, these data show that protein is not a major
regulator of mammary grth in rapidly-grown prepubertal heifers provided protein is
adequate for structural growth.

How might this lack of effect be explained? First of all I suggest that it can not be
explained by differences in laboratory techniques. Differences in techniques exists in the
literature for example, Stelwagen and Grieve (1990) measured total mammary DNA
instead of DNA in parenchyma. The current study used laboratory techniques similar to
Sejrsen et al. (1982), Capuco et al. (1995), and Radcliff et al. (1997) to evaluate
mammary development. Thus, difference in laboratory techniques used to quantify
mammary development does not explain the lack of a treatment effect on mammary
growth in the present study.

Another factor that might be important in considering effects of dietary protein on
rapidly growing heifers is the actual rate of gain. Rate of prepubertal BW gain in the
present experiment was ~1200 g/d and typical of those associated with inhibition of
mammary development (Sejrsen et al., 1982; Harrison et al., 1983; Petitclerc et al., 1984;

Valentine et al., 1987; and Capuco et al., 1995) and subsequent milk production (Gardner

41
et al., 1977; Little and Kay, 1979; Valentine et al., 1987; Gardner et al., 1988; Peri et al.,

1993; Capuco et al., 1995; and Van Amburgh et al., 1998). Therefore, my model was
well suited to test if increasing dietary protein from 14 to 19% alters mammary
development in rapidly-grown heifers.

Furthermore, the current study was conducted during the critical phase of
mammary development. The existence of a critical period for mammary development,
where grth of the secretory tissue is sensitive to high planes of nutrition, is widely
supported (Foldager and Sejrsen, 1987; and Johnsson, 1988). For example, Sejrsen and
coworkers (1982) reported that mammary development was less when prepubertal heifers
had ad libitum access to feed relative to restricted-fed heifers, but postpubertal mammary
development was the same regardless of feed intake. This sensitive period seems to
coincide with the allometric grth phase of the heifer mammary gland, which occurs
from 2 until 9 mo of age or shortly after puberty (Sinha and Tucker, 1969). In my
experiment, treatments were initiated at ~3.5 mo of age and continued for ~5.S mo.
Level of dietary CP had no effect on age at puberty, with the mean age of ﬁrst corpus
luteum being 7.5 months. All heifers were slaughtered at ~9 months of age. Therefore,
treatments were imposed during most of the critical allometric grth phase of the
mammary gland when impairment is normally seen.

In the current experiment mammary parenchymal DNA content at slaughter as a
measure of mammary development was ~1000 mg and BW at slaughter was ~320 kg. In
experiments by Sejrsen et al. (1982) and Capuco et al. (1995) mammary development of

rapidly-grown heifers relative to their controls was impaired ~30 and ~48%, respectively.

42
In both of these studies, heifers were killed at ~320 kg BW and rapidly-grown heifers

with impaired mammary development had ~1000 mg of parenchymal DNA. Comparing
mammary parenchymal DNA content in the present experiment to these previous studies
(Sejrsen et al., 1982 and Capuco et al., 1995) which used the same methodology for
measuring mammary parenchymal DNA indicates that impairment occurred in all
treatments. Therefore, results from this experiment dispute the theory that extra protein

will alleviate the detrimental effects of high energy on mammary development.

Hormones

Several studies have implicated somatotropin as a major mediator of dietary
effects on mammary development (Sejrsen et al., 1983; Johnsson et al., 1985; and Sejrsen
et al., 1986). Mammary growth is increased by exogenous somatotropin in lambs
(Johnsson et al., 1986; and McFadden et al., 1990), dairy heifers (Sejrsen, 1986; Sandles
and Peel, 1987; and Radcliff et al., 1997) and beef heifers (Glasser et al., 1991). Sejrsen
et al. (1983) reported that serum somatotropin concentrations were reduced in heifers
with ad libitum access to feed relative to restricted-fed heifers. Sejrsen also hypothesized
that decreased somatotropin concentrations were responsible for impaired mammary
parenchymal growth observed for heifers consuming a high-energy diet. Capuco and
coworkers (1995) reported that somatotropin concentrations and mammary development
were reduced ~25% and ~48%, respectively when rapid BW gains were achieved ﬁ'om
high intake of a corn silage-based diet but neither were reduced from high intake of an

alfalfa-based high protein diet. In the current experiment all heifers had ad libitum access

43
to their respective high-energy diet, and there was no difference in serum somatotropin

concentrations. Thus, results ﬁom the current experiment disagree with the idea that
higher protein may offset the reduction in somatotropin concentration caused by high-
energy diets. Therefore, one explanation for level of dietary protein not having an effect
on mammary development is that serum somatotropin concentrations were not different
among treatment groups.

In spite of its documented effect on mammary growth, the mechanism of action of
somatotropin is not clear. Although somatotropin receptor mRN A as been detected in
mammary tissue (Glimm et al., 1990), a direct effect of somatotropin on the mammary
gland is questionable. It has not been possible to demonstrate somatotropin binding to
mammary epithelial cells (Gertler et al., 1984; Akers, 1985; and Kazmer et al., 1986) and
somatotropin does not stimulate proliferation of isolated bovine epithelial cells (Collier et
al., 1993). Most evidence suggests that somatotropin does not act directly but rather it
acts indirectly on the mammary gland by other factors such as insulin-like growth factor-I
(IGF-I) (Akers, 1990).

Administration of somatotropin increases serum IGF-I concentrations (Bauman
and Vernon, 1993; Dahl, 1993; Purup et al., 1993a; Sharma et al., 1994; Vanderkooi et
al., 1995; Sharma et al., 1996; and Yung et al., 1996) and IGF-I receptors are present in
mammary tissue (Purup et al., 1995) and in isolated bovine mammary cells (Romagnolo
et al., 1994). Insulin-like-growth-factor-I also stimulates proliferation of isolated bovine
mammary epithelial cells (Zhao et al., 1992; Romagnolo et al., 1992 and Collier et al.,

1993). In the current study level of dietary CP did not affect serum IGF-I. Perhaps,

44
similar serum IGF-I concentrations not being different among treatment groups is another

explanation for the lack of a treatment effect on mammary development.

External measure of mammary development

There is a high correlation (r = 0.50 to 0.85) between mammary epithelial cell
number and milk yield, suggesting that increased mammary epithelial cell number results
in increased milk yield (Tucker, 1969). However, the terminal nature of mammary gland
dissection and analysis prevents researchers from extrapolating the effects of a treatment
on parenchymal grth to future milk production. Thus, it would be useful if there were
other methods of determining mammary development in young heifers so that the heifers
could be carried through lactation. Unfortunately, it is very difficult, if not impossible, to
get a reliable estimate of mammary grth in a live animal. Palpation of mammary
tissue has been used to assess mammary development in heifers, but this often gives
misleading results because the parenchyma constitutes a relatively small part of the total
mammary gland and parenchymal composition is not always constant (Sorensen et al.,
1964). Stelwagen and Grieve (1990) reported that morphometric evaluation of mammary
biopsies correlated poorly with chemical analysis of dissected heifer mammary glands.
The correlation coeﬁicient between ultrasonic area measurement and amount of
parenchymal DFF T is also poor (Niezen et al., 1996). However, the correlation
coefﬁcients (r) between measures of parenchymal tissue by dissection and computer

tomography were 0.80 for fat-free parenchyma and 0.62 for total parenchyma (Sorensen

45
et al., 1987). However, measuring parenchymal tissue by computer tomography is

expensive.

Teat length has been proposed as an additional parameter for assessing mammary
development in heifers (Moran et al., 1991). There are three advantages to measuring
teat length as a parameter for assessing mammary development: it is relatively easy, it is
not expensive, and it is non-terminal. There was a large variation in mammary
parenchymal mass (~200 to ~600 g/hemigland) and teat length (~20 to ~50 mm) in the
animals on the present study to test the relationship (Figure 3). In the current experiment,
variation in teat length accounted for only 1% of the variation in mammary parenchymal
mass. Thus, my results indicate that teat length is not a valid external estimate of

mammary development.

Growth and DMI

In the current experiment increasing dietary protein from 14 to 19% increased
mean BW gain and feed efﬁciency ~5% but the increase was not statistically signiﬁcant.
Previous experiments indicated that feed efficiency in heifers is improved as much as 7%
when dietary protein is increased from 11 to 16% (Bagg et al., 1985; and Heinrichs and
Lammers, 1998). Heinrichs and Lammers (1998) reported that DMI increased 2.6% and
BW gain increased 9.5% in rapidly-grown heifers fed 16% CP relative to heifers fed 12%
CP. Results ﬁ'om the current experiment seem inconsistent with those results because the

high level of dietary protein in my study did not alter DMI, BW gain or feed efﬁciency.

46
When evaluating heifer growth, an important parameter is skeletal growth. Body

weight as the sole criterion to deﬁne optimum size of an animal has limitations.
Markusfeld and Ezra (1993) demonstrated that withers height of Holstein replacement
heifers at ﬁrst calving was a better determinant of peak and 305-d ﬁrst lactation milk
yield than BW. An animal can be short and fat and weigh just as much as an animal that
is tall and thin. Skeletal size at parturition is important. Pelvic area is negatively
correlated to dystocia (Stevenson and Call, 1988). Thus, heifers that are short are more
likely to have increased incidence of dystocia. Heinrichs and Lammers (1998) reported
that increasing dietary CP ﬁom 11 to 16% increased the gain in hip height and width of
rapidly grown heifers 0.008 and 0.017 cm/d, respectively. In contrast, increasing dietary
CP ﬁom 14 to 16% did not inﬂuence indices of skeletal growth (Heinrichs and Lammers,
1998). In the current experiment, height at the withers and pelvic area at slaughter were
used to estimate skeletal size. In support of previous reports (Bagg et al., 1985; Kertz et
al., 1987 and Heinrichs and Lammers, 1998), the current study indicates that skeletal
growth is not improved in rapidly-grown dairy heifers by increasing dietary CP above
14%. Thus, feeding 19% instead of 14% CP to rapidly-grown prepubertal heifers did not
improve skeletal growth.

Higher-energy diets that produce accelerated BW gains increased body protein
deposition, but body protein deposition becomes proportionately less than that of fat
accretion (Radcliff et al., 1997 and Waldo et al., 1997). Previous studies suggest that
excessive fattening as a result of accelerated growth rates may increase incidence of

postpartum metabolic disorders (Grummer et al., 1995) and decrease ﬁrst lactation milk

47
yield (Hoffman et al., 1997). These problems have led investigators to seek replacement

heifer management strategies that increase grth without increasing body fat. One
strategy is to increase absorbed protein, which increases body protein accretion
(Hoffman, 1997). I was concerned that any effects of diet on carcass composition would
make it difﬁcult to interpret dietary effects on mammary development. However, Waldo
et al. (1997) reported that carcass composition was not different for rapidly-grown heifers
regardless of protein to energy ratio (54 or 83 g CP/Mcal ME) fed. Results from the
current study add support to Waldo et al. (1997) in that heifer BCS and carcass
composition were unaffected by the level of dietary CP. Therefore, our model was well
suited to test the idea that dietary protein affects mammary development of rapidly grown
heifers independent of effects on carcass composition.

In the current experiment, the correlation coefﬁcients between the percentage of
carcass protein, fat, and water estimated from the edible portion of the ninth-tenth-
eleventh-rib cut (Hankins and Howe, 1946) and from the edible portion of the half
carcass of 12 heifers were: r = 0.29, 0.71, and 0.46, respectively. In contrast, Hankins
and Howe (1946) found that correlation coefﬁcients between the percentage of carcass
protein, fat, and water estimated from the edible portion of the ninth-tenth-eleventh—rib
cut and from the edible portion of the dressed carcass were much higher: r = 0.94, 0.81,
and 0.94, respectively. The differences in correlation coefﬁcients may be the result of
fewer animals used and a more narrow range of protein, fat, and water in my experiment

than in Hankins and Howe (1946). Regardless, in the present study estimated

48
percentages of carcass protein, fat, and water were only 1 to 3 percentage points different

than the actual half carcass values.

Implications

The objective of feeding and management of replacement dairy heifers is to
produce the best possible cows. Success should not be measured solely in terms of
average rates of gain or feed efﬁciency but rather by the milk production potential of the
heifer once she becomes a cow. However, the period from birth to ﬁrst parturition is
expensive and many farmers have focused on trying to decrease the cost of rearing
heifers as a means to increase farm proﬁtability.

The most effective way to reduce rearing costs is to reduce the age at ﬁrst calving
without compromising BW at parturition. Age at onset of puberty is inversely related to
growth rate. Therefore, heifers need to be reared at accelerated grth rates (> 1000 g/d)
until puberty to obtain a substantial reduction of age at ﬁrst calving. Unfortunately, rapid
growth rates before puberty can have a negative inﬂuence on mammary grth and
future milk production.

Recent experiments suggest indirectly that type of diet or protein level can modify
the effect of accelerated grth rates on mammary development (Capuco et al., 1995,
Radcliff et al., 1997, VandeHaar, 1997; and Van Amburgh et al., 1998). The current
study, in which I directly examine the effect of dietary protein, indicates that high protein
perhaps could increase mammogenesis as much as 10% but the impairment of

mammogenesis may be as great as 30 to 50%. I strongly suggest that feeding high

49
dietary protein will not prevent the impaired prepubertal mammary development that

occurs in heifers fed for rates of gain > 1000 g/d. Therefore, decreasing the time to AFC
could increase proﬁts further but only if milk production is not impaired. To date, data

indicates this risk is not worth taking.

50

CONCLUSIONS

Level of dietary CP did not effect body growth, DMI or feed efficiency of heifers.
Height at the withers was similar for all treatments. Heifers fed the standard-protein diet
had greater pelvic area at slaughter than heifers fed the low or high-protein diets. Diet
did not alter total carcass protein and fat content as estimated by 9-10-11 rib section or
half carcass. The level of dietary CP did not affect age or weight at puberty. Serum
concentrations of somatotropin and IGF-1 were not inﬂuenced by diet. The major
objection to rearing dairy heifers at a high grth rate is compromised mammary
development and decreased subsequent milk production. Increasing dietary CP fed to
rapidly-grown prepubertal heifers from 14 to 19% produced 10% greater mammary
development but was not statistically signiﬁcant. I conclude that dietary CP does not
have a major effect on mammary development of rapidly grown prepubertal heifers.
Comparing measurements of mammary development in the present experiment to
previous studies indicates that impairment probably occurred; Thus, I suggest that
feeding high protein will not prevent the commonly observed impairment of mammary

development when prepubertal heifers are grown rapidly.

51

APPENDIX

52

APPENDIX

Table 8. The percentage of carcass lipid, protein, and water estimated from the three—rib
cut compared with the actual values from the right half carcass.

 

 

 

 

 

 

 

Estimated ﬁ'om three-rib cut Actual from right half carcass
Heifer 1D % Lipid % Protein % Water % Lipid % Protein % Water
1 20.3 Mv‘ 61.2 13.9 Mvl 66.4
2 21.2 18.1 61.9 23.5 17.0 62.7
6 18.6 18.1 60.1 18.7 19.5 60.1
11 20.1 17.8 60.6 15.6 18.5 60.1
13 16.9 18.1 57.9 13.4 19.1 67.5
17 19.9 17.9 60.2 19.4 20.5 68.2
20 18.3 18.6 59.2 17.7 18.2 63.6
21 16.8 19.5 57.5 18.8 20.0 60.6
23 15.3 18.8 56.1 11.4 20.1 58.9
30 26.5 16.4 64.0 23.7 18.7 68.1
53 25.1 16.3 62.9 21.0 17.0 66.6
56 21.5 17.9 62.1 17.1 , 18.7 62.3
Average 20.0 17.9 60.3 17.9 18.9 63.8

 

 

 

 

 

lMV = Missing value

 

53

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54

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