DIETARY GLUCOSE RESTRICTION, CHRONIC EXERCISE AND LITTER SIZE:
EFFECTS ON RAT MlLK AND MAMMARY GLAND COMPOSITIONS
April Y. Matsuno School of Dietetics and Human Nutrition
Macdonald Campus McGill University, Montreal
November, 1996
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of Master of Science.
8 April Y. Matsuno 1996
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ACKNOWLEDGMENTS
This thesis could not have been completad without the support and
assistance from many people. I would like to express rny sincerest thanks to my
supervisor Dr. K.G. Koski, for seeing something in me that I could not see in
myself, for her constant support both academically and personally and for
teaching me that I do not always have to believe what is in print! I would also
like to thank rny cornmittee members, Dr. E. Block and Dr. R.I. Cue for their
guidance and interest in my project. I would especially like to thank Dr. Cue for
his patience and the hours he spent discussing statistics with me. I am also
grateful to Agdhas Zamani, Susan Smith, Louise Lanoue and Sandra Miniaci
for their technical assistance and to Leslie Ann LaDuke, Anne Houston,
Francine Tardif and Lise Grant for their good humor and administrative help. I
would also like to thank Dr. R. Brousseau, Dr. L. Masson, Dr. R. J. Hemming and
the Biotechnology Research lnstitute of NRC for their technical advice and for
allowing me to use their facilities. I am eternally grateful to my husband, Al, who
endured a great deal of the 'fall-out' from the writing of this thesis with good
humor, and for al1 of his technical, domestic and emotional support. Finally, I
would like to thank my friends in the School of Dietetics and Human Nutrition,
past and present who made the past 2 years al1 the more memorable:
Susan Anderson Maryam Fotou hinia Cynthia Mannion
Tim Bayley Nancy Hanusaik Elise Mok
Ming Cha Tanya Howell Fady Ntanios
Caroline Crawford Giugetta lovino Andrea Papamandjark
Marco DiBuono Kathleen Lindhorst Shaila Rodrigues
Nancy Julien Xu-Jing Liu Jennifer White
ABSTRACT
Glucose is a principle precursor for milk lactose and de novo synthesis of
milk fat; therefore exercising during lactation could create competition for
glucose between exercising muscle and lactating mammary gland. This study
investigated the combined effects of materna1 dietary glucose (2076, 40%, 6O%),
exercise (chronically exercised, sedentary) and litter size (8, 12 pups) on rat
mammary gland composition, milk composition, milk yield and pup growth.
Chronic exercise increased milk fat concentrations and an interaction between
chronic exercise and 20% dietary glucose decreased milk lactose
concentrations compared to 40% or 60% glucose diets. Restricting materna1
dietary glucose also decreased 'milk fat concentrations and exercise decreased
rnamrnary fat. In addition, pups of dams fed the 40% glucose diet were heavier
on lactation day 15 than pups of dams fed the 60% diet. These results suggest
that competition for glucose occurs and that a 40% glucose matemal diet may
be more appropriate for pup growth.
iii
RÉSUMÉ
Le glucose est un pr6curseur principal de la synthèse du lactose et du
gras du lait; la practique d'exercices durant l'allaitement pourrait donc entrafner
une cornpetition pour le glucose entre les muscles au travail et la glande
mammaire. Cette recherche s'est intéressée aux effets combinés de la teneur
en glucose de la diète maternelle (20%, 40%, 60%), de I'exercice (exercice
chronique, sédentarité) et de la grosseur de la portée (8, 12 petits) sur la
composition de la gland mammaire du rat, la composition du lait, la production
de lait, ainsi que la croissance des petits. L'exercice chronique a augmenté la
concentration en matieres grasses du lait, et une interaction entre l'exercice
chronique et la diète contenant 20% de glucose a diminue la concentration en
lactose du lait contrairement aux diètes contenant 40% et 60% de glucose.
Limiter l'apport en glucose a diminué la concentration en matieres grasses du
lait et l'exercice a diminué le gras mammaire. De plus, les petits des femelles
nourries avec la diète contenant 40% de glucose étaient plus lourds au
quinzième jour d'allaitement que les petits des femelles nourries avec la diète à
60%. Ces résultats suggèrent qu'une compétition pour le glucose se produit et
qu'une diéte maternelle contenant 40% de glucose peut être plus appropriée
pour la croissance des petits.
TABLE OF CONTENTS
..................... .....*.........................*.*...............,,.... ACKNOWLEDGMENTS , ..,... i
. . LIST OF TABLES ..................................................................................................... 1/11
... LIST OF FIGURES ................................................................................................... vu1
LIST OF ABBREVIATIONS ................................................................................... x
INTRODUCTION AND STATEMENT OF PROBLEM ................................. 1
CHAPTER 1 ., REVIEW OF LITERATURE ..................................................... 3
I . EFFECT OF MATERNAL NUTRITION. EXERCISE AND LITTER SlZE ON MlLK COMPOSITION AND YlELD ........................... 3
A . Mammary Glucose Metabolism And Synthesis Of Milk Macronutrients (Lactose And Fat) ............................................................. 3
6 . Role Of Maternal Diet ................... ,...... ......................................................... 5 i) Energy Restriction ........................................................................................ 5
a . Animal Studies ................................................................................. 5 b . Human Studies ................................................................................ 8
ii) Protein Restriction ....................................................................................... 9 ................................................. iii) Dietary Carbohydrate/Fat Restriction 1 2
............................................................................ a . Animal Studies 1 2 ................................................................................ . b Human Studies 4
iv) Overnutrition .......................................................................................... . . 5
C . Impact Of Maternal Exercise On Milk Composition And Yïdd ......................................................................................................................... 16
..................... a . Animal Studies .. ..................................................... 16 ................................................................................ b . Human Studies 6
D . Impact Of Litter Sire On Milk Composition And Yield ..................... 17
II . EFFECT OF MATERNAL NUTRITION. EXERCISE AND LITTER SlZE ON MAMMARY GLAND GROWTH AND
................................................................................................. DEVELOPMENT 8
A . Mammary Growth And Development And Methods Of .................................................................................................... Measurement 18
B . Role Of Maternal Diet .................................................~.....~................b........b~... 22 i).Energy Restriction ..................................b....~..b........~....~.........................~...... 22 ii) Protein Restriction ....................................................................................... 24 ... III) Dietary CarbohydrateIFat Restrictions ................................................... 25 iv) Overnutrition .................... .. ..................................................................... 26
C . Impact Of Maternal Exercise On The Mammary Gland .................... 27
.................................... D . Impact Of Litter Sire On The Mammary Gland 28
III . CONCLUSION ................................................................................................. -30
IV . STATEMENT OF PURPOSE ......................................................................... 32 CHAPTER 2 - MATERIALS AND METHODS ............................................... 33
A . Experimental Design ........................................................................................ 33
B . Animal Care and Experimental Protocol ................................................ 33
C . Exercise Protocol .............................................................................................. 38
D . Experimental Diets ........................................................................................... 40
E . Quantitative Assays .......................................................................................... 42 i . Mammary Gland Composition .................................................................... 42 ii Milk Composition ........................................................................................ 44 ... IIL Plasma .......................................................................................................... 44
F . Data Management and Statistical Analyses ......................................... 45
................................................................................... CHAPTER 3 . RESULTS -48
A . Materna1 Food lntake ....................................................................................... 48
B . Maternal Body Weight ..................................................................................... 49
C . Plasma Analyses ............................................................................................... 51
D . Mammary Gland Composition ..................................................................... 52
E . Milk Composition ............................................................................................... 53
F . Pup Analyses ....................................................................................................... 54
.................................................... G . Correlation and Regression Analyses 56
H . Resuit Tables and Figures .......................................................................... 58
CHAPTER 4 - DISCUSSION ............................................................................... 94
BlBLlOGRAPHY ........................................................................................................ 1 14
APPENDlX 1 ............................................................................................................... 2 2
LIST OF TABLES
TABLE 2.1 Variables measured in the pregnancy and lactation .......................................................................... phases of the study. 35
TABLE 2.2 Composition of control and glucose (GLUC) restricted ................................................................................................... diets.. .4 1
TABLE 3.1 Cumulative maternal food intake (g) at the end of ................................................................... pregnancy and lactation. -5 8
TABLE 3.2 Repeated measures ANOVA of daily food intake during pregnancy and lactation: effects of dietary glucose level, chronic exercise, litter size and day. ................................................ 60
TABLE 3.3 Final maternal weight (g) at the end of pregnancy (prior to parturition) and at the end of lactation (Ld15). ............................... 64
TABLE 3.4 Repeated measures ANOVA of dam weight during pregnancy and lactation and 2-day weight gain during lactation: effects of dietary glucose level, chronic exercise, litter size and day. .............................................................. 66
TABLE 3.5 The effects of dietary glucose level, chronic exercise and ........................................................ c litter size on Ld15 dam plasma. 74
TABLE 3.6 The effects of dietary glucose level, chronic exercise and litter size on Ld15 mammary gland composition. .......................... 79
TABLE 3.7a The effects of dietary glucose level, chronic exercise and litter size on Ld15 milk composition, energy and yield ................. 83
TABLE 3.7b Maternai rnacronutrient intake (g/d) and milk rnacronutrient output (gld) on Ldl5 ............................................................................ 89
TABLE 3.8 The effect of maternal dietary glucose level, chronic exercise and litter size on individual pup weight, average daily milk volumelpup, average daily milk energy/pup and average daily milk fatlpup on Ld15. ......................................... 90
LIST OF FIGURES
FIGURE 2.1
FIGURE 3.1
FIGURE 3.2a
FIGURE 3.2b
FIGURE 3.2~
FIGURE 3.3
FIGURE 3.4a
FIGURE 3.4b
FIGURE 3 . 4 ~
FIGURE 3.4d
FIGURE 3.4e
FIGURE 3.4f
FIGURE 3.5a
Experimental design. .................................................................. ..34
The effect of dietaiy glucose level, chronic exercise and litter size on cumulative maternal food intake (g) at the end of pregnancy and lactation. ................................................ -59
The effect of dietary glucose level, chronic exercise and litter size on daily maternal food intake during
............................................................. pregnancy and lactation.. 6 1
The effect of dietary glucose level and chronic exercise on matemal food intake pattern during pregnancy. ................ 62
The effect of dietary glucose level, chronic exercise and litter size on maternal food intake pattern during
.......................................................................................... lactation. 63
The effect of dietary glucose level, chronic exercise and litter size on maternal weight (g) at the end of pregnancy (prior to parturition) and lactation (Ld15). ............. 65
The effect of dietary glucose level, chronic exercise and litter size on daily maternal weight during pregnancy
................................................................................... and lactation 68
The effect of dietary glucose level and chronic exercise .................. on matemal 2-day weight gain during pregnancy 69
Interaction effect between dietary glucose and chronic ........................................... exercise on dail y matemal weight.. -70
The effect of dietary glucose level and chronic exercise ......................... on matemal weight pattern during pregnancy 7 1
The effect of dietary glucose level and chronic exercise on 2-day maternal weight gain pattern during
...................................................................................... pregnancy .7 2
The effect of dietary glucose level, chronic exercise and litter site on materna1 weigM pattern during lactation ............. 73
The effect of dietary glucose levei, chronic exercise and litter size on Ldt5 dam plasma glucose and insulin -
............................................................................. concentrations.. .76
Abbreviations used throughout text:
BWtL = final lactation body weight
BWtP = final pregnancy body weight
CFeedL = cumulative food intake at the end of lactation
CFeedP = cumulative food intake at the end of pregnancy
d = day
D20 = 20% glucose diet
D40 = 40% glucose diet
D60 = 60% glucose diet
E = chronically exercised
ER = endoplasmic reticulum
Gd = gestation day
glm = general linear models
GLUC = glucose
hr = hour
Ld = lactation day
LDH = lactate dehydrogenase
LDL = low density lipoprotein
LPL = lipoprotein lipase
LSM = least square mean
MEpup = milk energy/pup
MFat = milk fat
MFatpup = milk fatlpup
MFGM = milk fat globule membrane
MG = mammary gland (refers to mammary tissue, not only glandular tissue
MGDNA = mammary gland DNA
MGFatD = mammary gland fat dry weight
MGFatW = mammary gland fat wet weight
MGGlyc = mammary gland glycogen
MGPro = mammary gland protein
MGWt = mammary gland wet weight
MGWtB = mammary gland wet weight as percent of body weight
min = minute
MLact = milk lactose
MPro = milk protein
MVolpup = milk volumelpup
MYield = milk yield
NEFA = non-esterified fatty acids
PCort = plasma cortisol
PGluc = plasma glucose
Plns = plasma insulin
PLDH = plasma lactose dehydrogenase
PNEFA = plasma non-esterified fatty acids
pupwtf = final pup weight (Ld15)
S = sedentary
SELSM = standard error of least square mean
Tkcald = total calorieslday
Tkcalml = total caloriesfml
VolPup = milk volumefpup
W = watts
wk = week
INTRODUCTION AND STATEMENT OF PROBLEM
Glucose is the principle metabolic fuel used by the fetus during gestation
and therefore adequate supply of glucose is critical for fetal growth and survival
(Koski and Hill, 1990). During the latter part of gestation, glucose is required to
build glycogen stores in fetal liver and heart tissue; deficits in these stores are
associated with increased perinatal mortality (Koski and Hill, 1986 and 1990).
Cornpetition for glucose between maternal muscle and fetus may be induced if
exercise is performed during gestation; previous studies have demonstrated
that maternal exercise decreases maternal and fetal liver glycogen (Gorski,
1983; Carlson et al., l986), increases maternal skeletal muscle glycogen
(Mottola and Christopher, 1991) and jeopardizes fetal glucose uptake
(Treadway and Young, 1989). Fetal weight, plasma glucose and insulin
concentrations as well as liver glycogen concentrations of offspring reportedly
decrease if inadequate maternal dietary carbohydrate is provided along with
acute maternal exercise (Cobrin and Koski, 1995).
Glucose is also considered the principle precursor for milk lactose and
de novo synthesis of milk fat (Williamson, 1980). It is therefore logical to
suspect that similar to exercising muscle and the neonatal system, cornpetition
for glucose may also occur between exercising muscle and the mammary
gland. Little research, however, has focused on the consequences of matemal
exercise and altered glucose supply to the mammary gland during lactation.
Adequate carbohydrate or at least a source of glucose equivalents has been
detemined to be necessary during lactation in non-exercising animals for the
synthesis of milk lactose and lipid (Koski and Hill, 1990; Koski et al., 1990).
However, only three studies (Treadway and Lederman, 1986; Lovelady et al.,
1990; Dewey et al., 1994) have investigated whether cornpetition for glucose
exists between exercising muscle and the lactating marnmary gland. One study
reported that milk lactose concentration decreased with exercise in rats and
attributed this to decreased glucose availability to the mammary gland due to
the energy requirement of exercise. The other two studies found no adverse
effects of exercise on human lactation performance. None of these studies,
however, examined the effects of altered diet or number of offspring in addition
to the exercise component. There are no published studies on the combined
effects of matemal diet, exercise and litter size on lactation performance. The
purpose of this study, therefore, was to investigate the combined effects of
maternal dietary glucose restriction, chronic exercise and litter size on lactation
performance.
The following chapter provides background information on the mammary
gland and the synthesis of milk macronutrients, and summarizes what is
currently known about the independent effects of maternal diet, exercise and
litter size on mammary gland growth and developrnent and milk composition
and yield.
CHAPTER 1 - REVIEW OF LITERATURE
1. EFFECT OF MATERNAL NUTRITION, EXERCISE AND LITTER
SlZE ON MlLK COMPOSITION AND YIELD
A. Mammary Glucose Metabolism and Synthesis of Milk
Macronutrients (Lactose and Fat)
Glucose is used by the mammary gland as both a fuel and a biosynthetic
precursor for milk lactose and a large portion of milk fat (Kuhn, 1978;
Williamson, 1986). In the rat, 10-20% of glucose absorbed by the rnammary
gland is used for lactose synthesis (Kuhn, 1978), and 60% is used for glycolysis
for cellular metabolism to provide triose phosphates for glycerol formation and
to provide carbons for acetyl CoA for eventual fatty acid synthesis (Kuhn, 1978;
Larson, 1985). The pentose phosphate cycle uses the remaining 20-30% of
absorbed glucose for generation of NADPH for lipogenesis (Kuhn, 1978).
The process of lactation can use up to 8040% of the materna1 body pool
of glucose since a continuous supply of glucose to the mamrnary gland is
required for synthesis of milk lactose and de novo milk fat (Kuhn, 1978). Any
decrease in the availability of glucose as would occur with a carbohydrate
restricted diet, would decrease the mamrnary gland's glucose uptake in order
for the lactating dam to presewe euglycemia (Williamson, 1986). Decreased
glucose uptake by the mammary gland can therefore affect milk composition
and yield . In the mamrnary gland (specifically the mammocyte), the final step in the
synthesis of lactose frorn UDP galactose and glucose is catalyzed by an
enzyme found on the inner surface of the golgi membrane called galactosyl
trançferase (Dils, 1989). Galactosyl transferase has a low affinity for glucose;
however, its affinity increases approximately 500-fold with transient
associations with a whey protein called alpha-lactalbumin (Mepham, 1987; Dils,
1989). Together, the enzyme and the whey protein are known as lactose
synthetase. It is suggested that the glucose molecule, a monosaccharide, is
able to pass through the golgi membrane via water-filled pores, but lactose, a
larger disaccharide, is unable to pass through these pores and thus
accumulates in the golgi lumen (Mepham, 1987). The accumulation of lactose
plays a crucial role in the flux of water from the cytosol to the golgi lumen and is
thus a major osmotic regulator of milk since most milk is approximately 80%
water (Mepham, 1987; Dils, 1989). Lactose is released from the golgi
membrane into the milk through the apical side of the mammocyte by exocytosis
(Dils, 1 989).
The major form of milk fat is triglyceride with the remaining milk fat
contributed by the milk fat globule membrane (MFGM). There are generally two
sources of precursors that make up the triglycerides; the first is blood lipids and
the second is glucose. With the help of lipoprotein lipase (LPL) secreted at the
capillary wall, the mammocyte extracts fatty acids from chylomicrons and low
density lipoproteins (LDL) in the blood. Fatty acids 2C18 and most Cl6 fatty
acids are derived from the blood. De novo fatty acids up to Ci6 in length are
generated from acetyl CoA molecules formed from glucose. The glycerol
rnoieties of triglycerides are provided by glucose (via glycolysis), free glycerol in
blood, and blood lipids following lipolysis as described above. The final step of
glycerol esterification to triglyceride occurs at the endoplasmic reticulum (ER)
membrane and requires fatty acid activation by ATP-dependent acyl-CoA
synthetase. Fat droplets (liposomes) pinch off from the ER and can fuse with
one another to form larger liposomes which move toward the apical surface of
the mammocyte and are released by exocytosis. These liposomes or fat
globules are surrounded by MFGM (Mepham, 1987).
B. Role Of Materna1 Diet
The effects of maternal diet manipulations such as energy restriction,
protein restriction, carbohydratelfat restriction and overfeeding on milk
composition and yield are presented below.
i) Energy Restriction
Several animal studies have investigated the effect of maternal dietary
energy restriction on milk composition and yield (Crnic and Chase, 1978;
Warman and Rasmussen, 1983; Roberts et al., 1985; Kliewer and Rasmussen,
1987; Grigor et al, 1987; Nicholas and Hartmann, 1991; Sadurskis et al., 1991);
however, fewer human studies have been published (Vuori et al., 1982; Strode
et al., 1986; Dusdieker et al, 1994). For clarity, the review of animal studies will
be divided into the major milk components (protein, lactose, fat) and milk yield.
a. Animal Studies
MlLK PROTEIN
With the exception of one study (Waman and Rasmussen, 1983), the
majority of evidence indicates that milk protein is not affected by maternal
dietary energy restriction. Studies have shown that milk protein concentrations
are not affected by matemal energy restriction in baboons (Roberts et al., 1985)
and rats (Cmic and Chase, 1978; Kliewer and Rasmussen, 1987; Grigor et al.,
1 987; Nicholas and Hartmann, 1991 ; Sadurskis et al., 1 991). Restrictions
varying from 40% to 79% of ad libitum intake during gestation andlor lactation
have not affected milk protein concentrations (Roberts et al., 1985; Kliewer and
Rasmussen, 1987; Grigor et al., 1987; Nicholas and Hartmann, l99l). A study
by Warman and Rasmussen (1983), however, showed milk protein to increase
significantly when rats were fed 50% of ad libitum intake both during gestation
and lactation and during lactation only. The authon offered no explanation for
the increase in milk protein. Given that similar duration of dietary restriction,
degree of restriction (Roberts et al.; 1985, Kliewer and Rasmussen, 1987; Grigor
et al., 1987), diet fed (Kliewer and Rasmussen, 1987) and rat strain (Kliewer
and Rasmussen, 1987) were used, an explanation for the significant effect on
milk protein is not apparent.
MILK LACTOSE
The effects of materna1 dietary energy restriction on milk lactose
concentrations (Warman and Rasmussen, 1983; Roberts et al., 1985; Kliewer
and Rasmussen, 1987; Grigor et al, 1987; Nicholas and Hartmann, 1991 ;
Sadurskis et al., 1991) are more varied than the effects on protein
concentrations. Energy restriction to 50% of ad libitum intake during gestation
and lactation or during lactation only decreased milk lactose levels in rats
compared to those fed ad libitum (Warman and Rasmussen, 1983; Kliewer and
Rasmussen, 1987); however, a 50% restriction in intake during gestation only
did not affect milk lactose concentrations at lactation day 14 (Kliewer and
Rasmussen, 1987). In comparison to the studies just described, other studies
employing more severe (40% ad libitum during lactation - Grigor et al., 1987)
and less severe restrictions (60%, 70%, 79% ad libitum during gestation and
lactation or lactation only - Roberts et al., 1985; Grigor et al., 1987; Nicholas and
Hartmann, 1991; Sadurskis et al., 1991) showed no differences in milk lactose
concentrations. The literature suggests that dietary energy restriction of 50%
either during gestation and lactation or during lactation only results in
decreased milk lactose levels, and less severe restrictions do not affect milk
lactose.
MlLK FAT
The effect of energy restriction on milk fat concentrations appears to be
inconsistent. Waman and Rasmussen (1983) found that 50% energy restriction
during lactation or during gestation and lactation, increased milk fat
percentages. The same energy restriction increased milk fat concentrations
after restriction du ring gestation only, du ring gestation and lactation, but not
during lactation only (Kliewer and Rasmussen, 1987). Energy restriction to 79%
of ad libitum intake decreased milk fat concentration when imposed during
gestation and lactation (Nicholas and Hartmann, 1991). Other studies imposing
energy restriction to 40°/0 (Crnic and Chase, 1978; Grigor et al., l987), 60%
(Roberts et al., 1985; Grigor et al, 1987) and 70% (Sadurskis et al., 1991) ad
libitum intake found no impact on milk fat (Crnic and Chase, 1978; Roberts et
al., 1985; Grigor et al., 1987; Sadurskis et al., 1991).
MlLK YIELD
With varying methods for detemining milk production (tritiated water and
calculation with litter weight gain), matemal dietary energy restriction appears to
result in decreased milk yield even with restriction to 70°h of ad libitum intake.
a Restrictions to 70% and to as low as 40% of ad libitum intake have decreased
milk yield in baboons (40% ad libitum intake-Roberts et al., 1985) and rats (70%
ad libitum intakeBadurskis et al., 1991; 40% and 60% ad libitum intake-Grigor
et al., 1987; 40%, 50% and 75% ad libitum intake-Young and Rasmussen,
1985). Milk yield per gram of materna1 body weight was decreased for rats
which were fed ad libitum pre-weaning (i.e. in utero and dunng suckling their
mothers ate ad libitum), but were food restricted (50% of ad libitum) after
weaning (Le. pre-pregnancy, pregnancy and lactation) (McGuire et al., 1995).
Milk production was not decreased in rats which experienced pre-weaning food
restriction and post-weaning free access to food. The authors speculated that
free access to food post-weaning alleviated the adverse effects of pre-weaning
food restriction. More specifically, post-weaning food restriction may not allow
adequate adipose tissue deposition, thereby limiting the extent to which
mammary ducts can develop. Milk yield thus suffers in the first lactation
(McGuire et al., 1995).
b. Human Studies
The human studies that have investigated the effect of maternal dietary
restriction on lactation agree that moderate energy restriction is not deleterious.
Given the ethical concerns with studying energy restriction (dieting) during
human lactation and its effects on breast milk composition and yield, few studies
have been published in this area. Two studies recruited well-nourished
volunteers to reduce their caloric intake during lactation (Strode et al., 1986;
Dusdieker et al., 1994) Twenty two women in a study by Strode et al. (1 986)
volunteered to be in the experimental or control group. The experimental group
reduced caloric intake by 20930% for one week, while the control group
maintained their intakes (regular intakes were determined in a one week base-
line phase). There were no significant differences between the two groups in
milk concentrations of lactose, protein and fat as well as milk output as
measured by infant intake. The authors concluded that lower maternal dietary
energy intakes may allow successful lactation as well as moderate maternal
weight loss, however, longer term studies were required. Dusdieker et al.
(1994) conducted a longer study (1 0 weeks) where 33 well-nourished wornen
who seived as their own controls decreased their energy intakes to -77% of
their baseline intakes. No significant differences in mean milk fat % or nitmgen
concentration were found at baseline and at week 10. Although milk yield
increased from 759 +_ 142 mUd at baseline to 802 + 189 mUd at the end of
week 10 it was unclear if this difference was significant. In agreement with
Strode et al. (1986), Dusdieker et al. (1994) concluded that moderate matemal
weight loss during lactation did not adversely affect milk quality or quantity.
A Finnish study examined the habitua1 post-partum diets of mothers at 6-
8 weeks post parturn and 17-22 weeks post parturn. Mean energy intake at 6-8
weeks was 9.8 MJ and at 17-22 weeks was 8.6 MJ. It is unclear if these energy
intakes were statistically different; however, no difference was found in total fat
content of breast milk sampled during these two time periods (Vuori et al.,
1 982).
Surnmary
The few human studies on the effect of materna1 energy restriction on
milk composition and yield agree that moderate energy restriction is not
detrimental to lactation. In addition, with the exception of milk fat, the animal
literature has revealed some trends of the effect of energy restriction on milk
composition. In animals, milk fat has been shown to increase, decrease or
remain unchanged under many levels of energy restriction and the literature did
not discuss in any detail possible reasons for or mechanisms of these
conflicting results. Lactose, on the other hand, appears to be stable until the
energy restriction has reached 50% of ad libitum intake where it generally
decreases, although one study's results did not agree with this trend. Of al1 the
milk macronutrients, protein seems to be the most stable under insult of energy
restriction. In addition, milk yield appears to decrease with even a relatively
mild energy restriction to 70% of ad libitum intake.
ii) Protein Restriction
For ethical reasons, only animal studies have investigated the effect of
protein restriction on milk composition and yield. In addition to level of protein
fed (Mueller and Cox, 1946; Crnic and Chase, 1978; Mansaray and Grimble,
1984; Grigor et al, 1987; Pine et al., 1994). protein quality has also been tested
(Mansaray and Grimble, 1984). As in the previous section on dietary energy
restriction, this section will be divided into the major milk macronutrients and
milk yield.
MlLK PROTEIN
Effects of dietary protein restriction on rat milk protein concentrations
have been shown to be as inconsistent as energy restriction on milk fat. In a
study by Mueller and Cox (1946), materna1 dietary restrictions to 5% and 10%
protein as casein did not affect milk protein concentration . Grigor et al. (1 987)
found that a 10% casein diet also did not change milk protein concentrations;
however, restriction to 9% casein decreased milk protein concentrations on
days 4 and 12 of lactation, but not on day 8 (Pine et al., 1994). Restriction to 80h
casein also decreased milk protein concentrations (Crnic and Chase, 1978). In
summary, milk protein levels have been shown to either decrease or remain
unchanged when dams have been subjected to protein intakes of 5-10%. Type
of protein or day of sampling cannot explain the discrepancy as casein was the
protein source used in al1 the studies and similar sampling days were used.
MILK LACTOSE
Variable results have been reported on the effect of protein restriction on
rat milk composition and yield. Restrictions during lactation to < 10% dietary
protein provided as casein have been shown to decrease (Mansaray and
Grimble, 1984; Pine et al., 1994) or to have no effect (Grigor et al., 1987; Pine et
al., 1994) on milk lactose concentrations depending on day of milk analysis.
Specifically, a restriction to 10% casein did not significantly affect milk lactose
concentration in day 14 milk (Grigor et al., 1987); however, a restriction to 9%
casein was found to decrease lactose concentrations on days 8 and 12 of
lactation, but not on day 4. Pine et al. (1 994) suggested that a protein deficit
may have limited the activity of lactose synthetase. A diet fed to rats containing
60 g milk proteidkg diet (-6%) resulted in significantly lower milk lactose
concentrations compared to a diet with 200 g milk proteinlkg diet (-20%). Diets
of 200 g (20%) and 100 g (10%) cereal proteintkg diet also decreased milk
lactose concentrations in cornparison to dietary protein provided as milk protein
(200 gtkg). A decrease in lactose synthetase activity was also highly correlated
with the decrease in lactose (r=0.794, p<0.001) suggesting that dietary protein
quality and quantity affect milk lactose via this enzyme (Mansaray and Grimble,
1 984).
MILK FAT
Protein restrictions have been shown to increase or to have no effect on
milk fat levels. Restrictions to 8% (Crnic and Chase, 1978) and 9% (Pine et al.,
1994) casein were shown to increase rat milk fat concentrations. In a study by
Pine et al., (1 994) milk fat was increased on days 4 and 8, but not on day 12.
Crnic and Chase (1978) found that an 8% casein diet increased milk fat on day
21 of lactation. Grigor et al. (1987) found no difference in milk fat from rats fed
10% casein vs. 20% casein diets at day 14 of lactation. It is not clear why milk
fat was increased in some studies, but not in others. Possible explanations
given by Pine et al. (1994) for an increase in milk fat could be a decrease in milk
volume without any alteration to milk fat synthesis or compensation for milk
energy lost by a decrease in milk protein.
MILK Y1EL.D
Three studies that investigated the effect of materna1 protein restriction,
measured rat milk yield and found that milk yield was decreased (Mueller and
Cox, 1946; Mansaray and Grimble, 1984; Grigor et al., 1987). Protein restriction
to 5% (Mueller and Cox, 1946) or 10% (Mueller and Cox, 1946; Grigor et al.,
1987) resulted in decreased milk yield. Mansaray and Grimble (1984) also
found a decrease in milk yield when 6% or 10% milk protein and 10% or 20%
cereal protein were fed compared to 20% milk protein. When compared to pair-
fed animals (200 gikg diet), feeding 6% milk protein and 20% and 10% cereal
protein were found to decrease milk yield (Mansaray and Grimble, 1984).
Summary
Materna1 dietary protein restriction was not shown to have consistent
effects on the milk rnacronutrients. Milk protein was decreased or unchanged
and milk fat was increased or unchanged. It is unclear why the protein levels
were decreased in some studies and not in others, given that similar levels and
qualities of protein were used. Feeding lower quality protein even in quantities
as high as 20% resulted in lower lactose levels. A possible mechanism of
action for protein quality and quantity is the limiting of lactose synthetase
enzyme activity.
iii) Dietary CarbohydratelFat Restriction
With respect to lactation, carbohydratelfat restriction appears to be the
least investigated area. However, unlike studies on protein restriction, both
animal (Grigor and Warren, 1980; Romsos et al., 1981 ; Koski et al., 1990; Koski
and Hill, 1990; Lanoue and Koski, 1994) and human studies (Harzer et al.,
1984; Silber et al., 1988) have been published.
a. Animal Studies
A range of materna1 dietary carbohydrate levels frorn O% to 60% and
their effects on milk composition have been investigated (Grigor and Warren,
1980; Romsos et al., 1981 ; Koski et al., 1990; Koski and Hill, I W O ; Lanoue and
Koski, 1994). Rats fed 0% glucose, fatty acid based diets (Koski et al., 1990)
and 4% glucose, fatty acid based diets (Koski and Hill, 1990) throughout
gestation (and lactation) did not produce milk. Use of fatty acid based diets
using oleic acid which eliminated a source of glucose equivalents contrîbuted
by glycerol, demonstrated the necessity for a minimum level of carbohydrate for
successful lactation. Romsos et al. (1981) also fed a O% carbohydrate diet to
dogs from 3 weeks post-conception to week 4 of lactation. The dogs were able
to lactate, and milk lactose and fat concentrations decreased and increased
respectively, but there were no changes in milk protein or total energy content
(Romsos et al., 1981). To explain why the dogs were able to lactate on a 0%
carbohydrate diet, Romsos et al. (1981) stated that during lactation dogs have
relatively low glucose requirements as their milk has a low lactose
concentration in cornparison to cow and human milk (Larson, 1978 cited in
Romsos et al., 1 981).
MILK PROTEiN
As found in Romsos et a1.k (1981) study using dogs, milk protein was not
significantly altered by a 6% glucose, fatty acid based diet (Koski et al., 1990), a
070 glucose, 4% lipid glycerol diet (Koski and Hill, 1990) or a 12% or a 24%
glucose diet (Lanoue and Koski, 1994) fed to rats throughout gestation and
lactation. Milk protein was also not altered with fatty acid based diets providing
4% glucose fed either during gestation or lactation only (Koski and Hill, 1990);
however, it was shown to increase when a 0% glucose, 4% lipid glycerol diet
was fed during lactation only (Koski and Hill, 1990).
MILK CARBOHYDRA TE
Milk carbohydrate decreased when either 6% glucose (Koski et al., 1990)
or 0% glucose, 4% lipid glycerol diets (Koski and Hill, 1990) were fed
throughout gestation and lactation. Milk carbohydrate was also decreased
when 0% glucose, 4% lipid glycerol diet was fed during lactation only (Koski
and Hill, 1990). A fatty acid based, 4% glucose diet fed during gestation only,
increased milk carbohydrate, but when fed during lactation only, milk
carbohydrate was not altered (Koski and Hill, 1990). Milk carbohydrate (lactose
and glucose) was also not altered by 12% or 24% glucose diets fed throughout
gestation and lactation (Lanoue and Koski , 1 994).
MlLK FAT
Milk fat has been shown to decrease with 6% glucose diets (Koski et al.,
1990), 0% glucose, 4% glycerol diets (Koski and Hill, 1990) and 12% or 24%
glucose diets (Lanoue and Koski, 1994) fed throughout gestation and lactation
compared to 60% glucose diets. Interestingly, restricted carbohydrate diets fed
either during gestation only (4% glucose, fatty acid based diet-Koski and Hill,
1990) or lactation only (0% glucose, 4% lipid glycerol or fatty acid based 4%
glucose diets-Koski and Hill, 1990) did not alter milk fat. Grigor and Warren
(1980) found that feeding diets with O%, 10% or 20% fat for 7 days during
lactation also did not change the milk fat concentrations of rats.
MLK ENERGY
The majority of variations in milk macronutrients appear to equalize in
terrns of total milk energy as total milk energy did not change significantly when
a Ooh carbohydrate diet was fed during gestation and lactation (Romsos et al.,
1982)) a 0% glucose, 4% lipid glycerol diet was fed during lactation only or a
4% glucose, fatty acid based diet was fed during gestation only or lactation only
(Koski and Hill. 1990). A 6% glucose and 0% glucose, 4% lipid glycerol diet fed
throughout gestation and lactation, however, did decrease metabolizable
energy of milk (Koski et al., 1990, Koski and Hill, 1990). This is not surprising
considering the decrease in milk carbohydrate and fat which resulted from the
use of these diets.
b. Human Studies
Two human studies manipulated the proportions of dietary fat and
carbohydrate fed to lactating women. In a study which investigated the effects
of low fat/high carbohydrate and high fat/low carbohydrate diets on milk
composition, milk protein did not change; however, milk lactose and fat were
affected (Harzer et al., 1984). Milk lactose concentration from milk of mothers
on a low fat/high carbohydrate diet was -213 of that of milk from mothers on a
high fatnow carbohydrate diet. The high fatllow carbohydrate diet resulted in
higher milk triglycerides. The study results should be interpreted with caution
as the study was comprised of only three subjects and the period of study was
short at only 2 weeks. In addition, the data was not analyzed statistically given
the small sample size.
Another human study employed a low fatlhigh carbohydrate diet for 5
days (5% fat, 15% protein, 80% carbohydrate) during the lactation period
(Silber et al., 1988). Women who gave birth at term (n=5) or prematurely (n=5)
had similar responses to the diet with respect to milk composition. Total fat and
nitrogen concentrations were not changed between days O and 4 of the diet
period; however, lactose concentrations increased and energy content
decreased significantly (Silber et al., 1988).
Summary
The animal studies show that adequate carbohydrate (or at least a
source of glucose equivalents) is essential for nomal lactation performance. In
both animal and human studies milk protein appears to be the least affected by
dietary carbohydrate restriction. Low carbohydrate diets (5 24% glucose) fed
throughout gestation and lactation decreased milk fat; however, very low
carbohydrate diets fed either during gestation or lactation only did not alter milk
fat. Milk carbohydrate was decreased by very low carbohydrate diets fed
throughout gestation and lactation, but not by 12% or 24% glucose diets.
iv) Overnutrition
Boyd et al. (1978) investigated the effect on milk fat of the addition of
excess energy as tallow or cornstarch to the diets of gilts and sows just pnor to
parturition and during lactation. Tallow and cornstarch were added and other
diet components adjusted such that the diets contained -3550 kcal more than
the control diet. Addition of tallow to the diets during gestation increased the
milk fat in colostrum, but this increase was not sustained. Addition of tallow
during lactation increased milk fat for the entire lactation period in cornparison
to controls.
C. Impact Of Maternal Exercise On Milk Composition And Yield
60th animal (Karasawa et al., 1981 and Treadway and Lederman, 1986)
and human studies (Lovelady et al., 1990, Dewey et al., 1994) which have
examined the effect of exercise on lactation have noted no significant effects of
exercise on milk yield.
a. Animal Studies
Only one animal study has been identified that examined the effect of
exercise throughout pregnancy and lactation on milk composition. Treadway
and Lederman (1986) trained Wistar rats for 7 weeks prior to mating to swim 2
hrld X 5 dfwk with a 3% tail weight. Analysis of day 15 milk showed significantly
lower amounts of lactose in milk from exercised rats. Milk energy, however, was
not statisticaliy different between the groups.
A study of voluntary wheel running throughout pregnancy and lactation
(Karasawa, 1981) found no significant change in milk yield per day in exercised
and non-exercised mice.
b. Human Studies
There have been two published studies on women which investigated
the effect of aerobic exercise on lactation (Lovelady et al., 1990 and Dewey et
al., 1994). The study by Lovelady et al. (1990) used self-selected subjects to
either exercise at 70% of maximal heart rate (MHR) 2 45 rninld X 5 d/wk or
remain sedentary. No differences were found with respect to milk energy, fat,
protein or lactose content. The second study done by the same group (Dewey
et al., 1994) tried to address some criticisms of the first study by conducting a
randomized trial of the effect of materna1 aerobic exercise on lactation. Again,
no significant differences were found in the volume or composition of the breast
milk.
D. Impact Of Litter Size On Milk Composition And Yield
Studies using rats (Morag et al., 1975, Yagil et al., 1976, Russel, 1980,
Fiorotto et al., 1991) and mice (Knight et al., 1986) have consistently shown
increased milk yields from dams nursing large litters vs. small litters. In addition,
litter size does not seem to affect milk lactose 'concentrations; however, the
effect of litter size on milk fat and protein concentrations has not been
adequately studied.
MlLK PROTEIN
Yagil et al. (1976) and Fiorotto et al. (1991) found decreased milk protein
concentrations from dams that nursed smaller litters compared to larger Mers (2
or 5 vs. 611 011 6-Yagil et al., 1976; Fiorotto et al., 1991) However, Yagil et al.
(1976) found no effect on milk protein when Mers of >6 pups were nursed.
Grigor et al. (1986) also showed no effect of litter size (2 pups vs. 10 pups) on
milk protein.
MILK LACTOSE
The two studies reviewed that measured the impact of litter size on
lactose concentrations agree that lactose concentrations remain unchanged
whether dams nurse srnall(2 or 4 pups) or large (1 0 or 16 pups) litters (Grigor et
al., 1986; Fiorotto et al., 1991).
MlLK FAT
Yagil et al. (1976) and Fiorotto et al. (1991) both found increased milk fat
if dams nursed smaller litters (2 vs. 6 and 10, and 4 and 10 vs. 16 respectively).
However, Fiorotto et al. (1991) found this to be true only after 6 days of lactation.
At 4 days of lactation, no difference was found in milk fat among litter size
groups. Milk fat was also the same for litter groups of 6 and 10 pups in the study
by Yagil et al. (1976). Grigor et al. (1986) found no difference in milk fat
concentrations between 2 and 10 pup litter size groups.
II. EFFECT OF MATERNAL NUTRITION, EXERCISE AND LITTER
SlZE ON MAMMARY GLAND GROWTH AND DEVELOPMENT
A. Mammary Growth and Development and Methods of
Measurement
The following sections on mamrnary gland growth and developrnent are
intended to give the reader an adequate background with which to understand
the literature review. Many other factors such as blood supply, hormonal control
and neural stimuli also influence the mammary gland and the milk it produces
and are reviewed elsewhere (Mepham, 1987; Martinet and Houdebine, 1993).
All female mammals from rodents to humans share the capacity to
transfer nutrition in the form of milk via the mammary glands from mother to
offspring. Possession of mammary glands is a distinguishing characteristic that
separates mammals from the rest of the animal kingdom and despite
differences in mammary size, number and anatomical position across species,
mammary glands are histologically very similar (Mepham, 1987). Development
of the mammary gland begins early on in ontogenesis even before the species
of the embryo can be identified (Larson, 1985). Regardless of species, the
embryo progresses through several identifiable stages of ectoderrnal thickening
ending with the mammary bud frorn which ail mammary glands originate. The
mammary bud continues development into a branch called a primary sprout. As
proliferation of the primaty sprout continues, canalization occurs where cells
proliferate at the surface to form rudimentary teats which will eventually allow
milk to exit the mammary gland. The number of these openings or
galactophores varies from species to species with mice and rats having one
opening per teat and humans having as many as 15-25 openings per teat. The
canal in the center of the primary sprout will eventually becorne the gland
cistem at the proximal end and the teat cistem at the distal end of the sprout. In
some species, branches of the primary sprouts, called secondary sprouts also
develop eventually becoming milk ducts draining into the gland cistern (Larson,
1 985).
Ectodermal cells give rise to the primary and secondary sprouts as just
described. Another cell layer of the embryo, the mesoderm, gives rise to
connective tissues (areolar, fibrous and elastic), adipose tissue and blood
(Larson, 1985). These tissues are also referred to as stromal tissue (Mepham,
1987). Development of adequate adipose tissue (fat pad) surrounding the
mammary bud is necessary for successful mammary gland growth (Larson,
1985) as it provides structural support and regulates the development of ductal
and secretory tissue (parenchyma - Mepham, 1987).
At birth, rats and mice are immature and helpless and their mammary
glands have not developed beyond the teat, teat cistern, gland cistern and
primary milk ducts. Growth of the mammary gland from birth to puberty (-40
days in rats) is isometric (Larson, 1985) with modest extension of the ducts into
the stromal fat pad. The fat pad extends slightly through cellular hypertrophy
(Mepham, 1987).
At the time of puberty, allometric growth of the mammary gland
commences just prior to onset of ovarian cycles (Mepham, 1983). The fernale
releases follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from
the anterior pituitary which causes the ovaries to synthesize and release
estrogen and progesterone. During each cycle, a surge in estrogen stimulates
mammary gland growth (mainly ductal lengthening and branching) (Larson,
1985).
In animals with short cycles (4-6 days in rats and mice), the functional
phase is follicular and the luteal phase occurs only with coitus or the presence
of other stimuli which both cause release of the luteotropic (in these species)
hormone prolactin (Larson, 1985). With each estrous cycle the milk ducts
lengthen and branch, but no secretory material (alveoli) deveiop until the
animal becomes pregnant (Larson, 1985).
Secretory tissue is made up of specialized epithelial cells which are
grouped into pear-like structures called alveoli. Groups of alveoli called lobules
drain into a common duct. This system is called the lobulo-alveolar systern and
as it grows it replaces the stroma1 fat pad (Mepham, 1987). Milk is secreted into
the lumen and drains into narrow ductules which in turn drain into larger ducts.
These larger ducts drain to a common reservoir (gland cistem) at the base of
the teat. Since this reservoir is small, most of the milk remains in the lobulo-
alveolar tissue. Myoepithelial cells surrounding the alveoli facilitate milk
removal. When stimulated to contract, milk is forced into the ducts.
Lobulo-alveolar growth begins slowly and increases as pregnancy
advances and is largely controlled by hormones. Chorionic gonadotrophin or
pregnant mare serum gonadotrophin (PMSG in animals; in humans the
hormone is human chorionic gonadotrophin), placental lactogen (PL) and
relaxin play pivotal roles. PMSG is secreted by the placenta into the blood and
has follicle stimulating capacity (Larson, 1985). Follicles are needed to produce
estrogen and progesterone for growth and maintenance of the uterus and
mammary glands. Towards the latter half of pregnancy, PMSG disappears
when the placenta is mature enough to produce sufficient amounts of estrogen
and progesterone. Placental lactogen, originally from the fetal placenta, acts
cooperatively with estrogen and progesterone to aid mammary gland growth
and may aid the growth of the uterus, placenta and fetus. Relaxin acts to soften
the connective tissues of the pelvis to allow enlargement of the birth canal and
the cervix to allow passage of the fetus (Larson, 1985).
After parturition, exponential growth of the mammary gland continues for
-5 days post partum and thus mammary DNA increases exponentially if
suckling of young is permitted (Larson, 1985; Mepham, 1987). The amount of
milk produced during lactation is largely dependent on the number of secretory
cells; however, other factors such as precu rson for milk synthesis, hormones
and frequent milk removal are also important factors. Once milk removal is
stopped (when the offspring are weaned), involution of the mammary secretory
tissue is accelerated with the increasing intra-marnmary pressure inhibiting any
fuither milk synthesis (Larson, 1985).
Macromeasures such as palpation and ruler measurements as well as
histological and cytological measures have been used to monitor mammary
gland growth and development. In vitro methods have also been used but with
limited success (Larson, 1985). In vivo techniques requiring surgical excision of
the mammary glands as used in the studies reviewed below allow growth and
development to be monitored through rneasurements such as MG weight as
well as protein, lipid, DNA and RNA concentrations.
B. Role Of Materna1 Diet
As with the section on milk composition and yield, the effects of materna1
dietary manipulations such as energy, protein and carbohydrate or fat restriction
on the mammary gland will be presented.
i) Energy Restriction
Several studies investigating the growth and development of mammary
glands have imposed energy intake restrictions on pregnant (Sykes et al., 1948;
Rosso et al., 1981; Mellor and Murray, 1985; Park et al., 1994) or lactating
animals (Grigor et al., 1987; Park et al., 1994). A study by Sykes et al. (1 948)
compared two groups of female white rats. Throughout gestation, group 1 was
fed stock diet ad libitum and group 2 was fed only 70% of the arnount fed to
group 1, but received the same amounts of protein and other nutrients. Lettuce
and raw carrots were supplemented to al1 animals weekly. At parturition, rats in
group 1 had mammary glands that were 80% heavier than those of group 2,
however, by day 21 of lactation, restricted animals fed ad libitum during
lactation had made up the weight difference found at parturition as mammary
gland weights were similar in both groups (Sykes et al., 1948).
A more severe energy restriction was used in a study by Rosso et al.
(1981). Sprague-Dawley rats fed 50% of ad libitum intake of a standard lab diet
from day 5 to day 21 of gestation had lower mammary gland weights, lower
mammary DNA and protein contents and a lower protein:DNA ratio compared to
control animals fed ad libitum (Rosso et al. 1981). It should be noted that al1
nutrients were restricted and that tissue results were analyzed as total mg as
opposed to concentration (mglg of MG tissue). Not correcting MG variables for
MG weight and mammary gland weight for body weight could have affected any
significant diff erences found.
In a study on ewes by Mellor and Murray (1985) it was shown that ewes
maintained on low planes of nutrition (Le. food quantities to maintain blood
glucose ranging from 1 . O 4 3 mmol/L; animais were thus undernourished) from
day 105 of gestation had a significantly lower mean udder weight at term
compared to ewes maintained on high planes of nutrition (Le. food quantities to
maintain blood glucose ranging from 2.8-3.3 mmolll).
More recently, Park et al. (1994) showed that rat mammary concentration
of protein at mid-pregnancy was significantly higher in Sprague-Dawley rats fed
70% of ad libitum intake compared to control rats. Dietary carbohydrate (-52%
cornstarch and sucrose vs. 65% in the control diet) was the only limiting
macronutrient in the restricted diet. When mammary protein concentrations
were averaged over mid-pregnancy, mid-lactation and involution, there was no
longer any significant difference between the two groups. DNA, lipid and
protein:DNA ratio were not significantly different at mid-pregnancy, mid-
lactation, involution or averaged over the three time periods. The less severe
energy restriction used in this study compared to the one used by Rosso et al.
(1981) as well as the fact that only energy as dietary carbohydrate was limiting,
could explain why significant differences in mammary gland outcornes were not
found in this study.
Grigor et al. (1987) restricted intake of Wistar rats to 40% and 60% of ad
libitum intake of a commercial pelleted diet from day 7 to day 14 of lactation.
Mammary glands of rats ied 40% and 60% of ad libitum intake were significantly
lower in weight than controls. Moreover, mammary glands of the 40% diet
group were significantly lower in weight than mammary glands from the 60%
diet. No differences in mammary DNA contents were found among these
groups (Grigor et al., 1987).
Summary
Energy restriction during gestation appears to decrease mammary gland
weight; however, catch-up growth is possible with re-feeding during the
lactation period. Inconsistent results have been reported regarding mammary
protein, DNA and cell size (PR0:DNA ratio). However, the decreased MG
weight, MG DNA, MG protein and protein:DNA ratio reported by Rosso et al.
(1981) may be explained by their use of a more severe energy restriction which
included restriction of protein, fat, vitamins and minerals and/or by not correcting
for mammary gland weight. Only one study reviewed examined the effects of
energy restriction during lactation and it appears to decrease mammary gland
weight in rats during this period was also reported (Grigor et al., 1987).
ii) Protein Restriction
Several studies have examined the effect of dietary protein restriction on
mammary gland weight (Pyska and Styczynski, 1979; Pau and Milner, 1982;
Sampson et al., 1986; Grigor et al., 1987; Pine et al., 1994). It was found that ad
libitum fed diets with restrictions to I 10% protein (9%-Pine et al., 1994; 10%-
Pyska and Styczynski, 1979; Grigor et al., l987), during gestation and lactation
or just during lactation significantly decreased mammary gland weight in
cornparison to control animals fed a diet with at least 15% casein (Pyska and
Styczynski, 1979; Grîgor et al. 1987; Pine et al. 1994). Restrictions to 10%
(Pyska and Styczynski, 1979) and 1 1 % protein (Sampson et al. 1986) during
gestation and lactation resulted in lower mammary DNA contents compared to
control animals fed 15% and 21 % protein diets respectively. Significantly lower
protein contents (total grams) were found in mammary glands on lactation days
8 and 12 after dietary protein restriction to 9% during gestation and lactation
(Pine et al., 1994). In addition, arginine deficient diets fed ad libitum with a level
of 14.2% protein during gestation resulted in significantly lower mammary gland
DNA content at gestation day 20 compared to control fed animals (Pau and
Milner, 1982).
Type of protein has also been shown to affect mammary gland growth. A
13% protein diet provided to rats as wheat gluten from conception to day 15 of
lactation was shown to decrease mamrnary gland weight, DNA and protein
concentrations in cornparison to non-purified laboratory chow diet (Jansen et
al., 1987).
Summary
Adequate feeding of protein to pregnant and tactating animals appears to
be crucial for normal mammary gland growth and developrnent as mammary
gland weight, protein andlor DNA contents were decreased by restriction of
dietary protein to $ 14.2% either during gestation or lactation or during both
gestation and lactation.
iii) Dietary CarbohydratelFat Restrictions
Only two studies have used low carbohydrate/high fat diets to examine
their effects on the mammary gland composition (Grigor et al., 1984; Lanoue
and Koski, 1994). Lanoue and Koski (1994) fed pregnant and lactating rats one
of four isocaloric diets with graded levels of glucose (0, 12, 24 and 60%).
Mammary gland cell size as estimated by protein:DNA ratio was decreased by
restriction of dietary glucose to 12% Mammary gland DNA concentration was
significantly greater in rats on the 12% glucose diet vs. 24% and 60% diets,
reflecting the decreased mammary cell size (protein:DNA ratio). No differences
were found in mammary gland protein, fat or glycogen concentrations.
In a study by Grigor et al. (1 984). the effects of chronic consumption of
what were classified by the authors as a high-fat diet (Le. restricted
carbohydrate) vs. a low-fat diet (Le. restricted fat) were examined over two
successive lactation periods. Mammary abdominal gland weights of rats fed the
high-fat (20%) diet were significantly greater than those of rats fed the low-fat
(4%) diet from the beginning of the second lactation period to the end of the
third lactation. Moreover, the mammary gland weights from both the high-fat
and low-fat diet groups were greater than those fed an undefined commercial
chow diet. Percentages of fat in the diets used by Lanoue and Koski (1994)
which ranged from 16% in the 60°' glucose diet to -40% in the 0% glucose diet
were higher than those used by Grigor et al. (1984) . In addition, even though
the diets used by Grigor et al. (1984) were presented as high fat (20% by weight
corn oil) the carbohydrate fraction, consisting of sucrose (30%) and starch
(25%), contributed 55% (by weight) as carbohydrate which is close to the 60%
glucose control diet used by Lanoue and Koski (1 994). No direct comparisons
between the two studies can be made; however, since mammary gland
concentrations of protein, fat and glycogen were not measured in the study by
Grigor et al. (1984) and mammary gland weight was not reported by Lanoue
and Koski f 1994).
Summary
Given the small number of studies and the lack of corroborating results
from each study, no strong conclusions can be drawn about the effects of a low
carbohydratelhigh fat diet on the mammary gland.
iv) Overnutrition
In a study aimed at detennining the effects of increased dietary energy
and protein or ovemutrition on mammary development, Weldon et al. (1991) fed
gilts either adequate dietary levels of energy or increased levels of dietary
energy and either adequate or increased levels of dietary protein during late
gestation. Mammary parenchymal weight, total DNA and total protein were
significantly greater for gilts fed adequate energy compared to energy over-fed
gilts. However, DNA, RNA, protein and lipid concentrations (Le. mgfg) as well
as RNA:DNA, total RNA and total lipids were not significantly different between
the groups. Over-feeding of protein did not significantly affect the mammary
gland variables mentioned above, thus it would appear that at least in gilts,
over-nutrition in terms of energy is detrimental to mammary gland development.
C. Impact Of Materna1 Exercise On The Mammary Gland
Only two studies have investigated the effect of materna1 exercise on
mammary gland growth and development (Karasawa et al., 1981; Mottola et al.,
1986). The small body of available literature does not clarify the effects of
exercise on mammary gland growth and development and demonstrates a
need for more studies in this area. Karasawa et al. (1981) examined the effect
of voluntary wheel running by mice throughout pregnancy and lactation on their
lactational performance. The precise amount of exercise that was done by the
mice was not reported. Running activity of about 5000 rotationdd in a 13.5 cm
diameter treadwheel at the beginning of gestation declined to about 500
rotationdd at term and remained at a low level during lactation. No differences
were found between the running group and a non-running control group with
respect to mammary gland wet weight, fat, protein and DNA contents. Given
that the rats ran voluntanly, the actual difference in exercise between the two
groups may not have been great enough to show a difference.
Mottola et al. (1986) studied the effects of strenuous exercise on a motor
driven treadmill (30 m/min X 120 minfd X 5 dfwk at a IO0 incline) during
gestation on the gross anatomy of the fernale rat. This amount and intensity of
exercise decreased the weight of the skin component which included mammary
tissue as well as skin, fur, subcutaneous tissue, fat and subscapular fat in
cornparison to pregnant sedentary controls. Given al1 the tissues included in
the skin component, it is difficult to attribut0 the difference to mammary gland
alone. The authors, however, speculated that the decreased weight could be
due to decreased deposits of materna1 subcutaneous fat due to increased lipid
oxidation (induced by exercise) or to underdevelopment of the rnammary gland
tissue.
D. Impact Of Litter Size On The Mammary Gland
In order to detemine if and to what extent offspring may control their own
destiny by influencing mammary gland growth and development of the dam,
several studies have investigated the effect of litter size on the mammary gland
(Nagasawa and Yanai, 1971; Tucker, 1966; Yagil et al., 1976; Wilde and Kuhn,
1979; Knight and Peaker, 1982; Grigor et al, 1986). In 1971, Nagasawa and
Yanai established a positive correlation between fetal number and mammary
gland development during gestation in mice. In addition, a correlation between
DNA content of rat mammary glands and the number of suckled young was also
reported (Tucker, 1966). In a study by Yagil et al. (1976), histological
examination of alveolar cells of dams that nursed 2, 6 or 10 pups did not reveal
any differences in the number of cells/alveolus, however, alveolar area
increased significantly as the litter size increased. lncreasing pressure in the
alveolar cells due to milk accumulation would explain the increase in alveolar
area (Yagil et al. 1976) as there is an increased milk requirement to feed larger
litters.
Wilde and Kuhn (1979), examined dams on day 12 of lactation and found
that mammary glands of rats that nuned larger litters (9-1 1 and 14-1 5 pups)
had more mammary tissue (fresh weight) than those that nursed smaller litten
(3-5 pups). The differences between the three groups were not proportionate to
the differences in the number of young nursed and it was unclear whether the
differences were statistically significant. The authors suggested that a greater
production of placental lactogen may have been responsible for the greater
rnarnmary tissue weight seen in dams that nursed larger Mers (Wilde and Kuhn,
1979). Placental lactogen as previously mentioned, is a mammogenic hormone
and is present in amounts proportional to the number of fetuses (Martinet and
Houdebine, 1993)
Knight and Peaker (1982) showed that the offspring of mice can
influence mammary gland development both pre- and post-partum. Mice were
hemihysterectomized during pregnancy to control the number of feto-placental
units and then were allowed to deliver their pups in order to study the effects of
litter size post-partum. Total DNA content of mammary glands was significantly
lower in hemihysterectomized mice compared to sham-operated control mice at
day 18 of gestation but not at day 13. In addition, they also found that at
lactation day 5 mammary glands of hemihysterectomized mouse dams who
nursed smaller Mers (4 pups) had lower mammary gland weights and
mammary DNA compared to sham-operated mice which nursed larger Mers (9
pups). lncreased placental lactogen production by dams pre-partum and
suckling-induced prolactin secretion post-partum are proposed mechanisms for
fetallneonatal control over mammary gland growth and development (Knight
and Peaker, 1982).
Grigor et al. (1 986), found highly significant differences in rat mammary
gland DNA concentrations when comparing dams that nursed 2 or 10 pups to
day 12-16 of lactation. Mammary gland DNA concentration of dams that nursed
10 pups was approximately 2.5 times the amount found in dams that nursed 2
pups, suggesting that epithelial cell proliferation was greater in the mammary
glands of dams that nursed larger litters.
Summary
The literature agrees that litter size does affect mammary gland growth
and development both pre- and post-partum. Larger litter sizes result in
increased mammary tissue weight, increased DNA concentration and increased
alveolar area.
III. CONCLUSION
Review of the literature revealed that some of the independent effects of
materna1 diet, matemal exercise and litter size on mammary gland growth and
development and on milk composition and yield have been studied more
adequately than others. Glucose is a principle precursor for milk lactose and de
novo synthesis of milk lipid and it has been demonstrated that adequate dietary
carbohydrate or a source of glucose equivalents is required for lactation to
occur. In addition, of al1 the milk macronutrients, protein appears to be the least
affected by dietary restrictions of energy and carbohydrate, and by matemal
exercise. Milk lactose and lipid seem to be more easily modified by dietary
restrictions, but the exact nature of these modifications has not been clearly
demonstrated. It is apparent that increased litter sizes lead to increased milk
yield; however, the effects of litter size on the milk macronutrients, especially
milk fat and protein require further investigation. Less has been published on
the effects of maternai diet, exercise and litter size on the lactating mammary
gland; however, it appears clear that dietary protein is required for mammary
gland growth and that increased litter size results in increased mammary gland
weight and DNA. However, in the areas of dietary carbohydrate and matemal
exercise and their effects on mammary gland growth and developrnent there is
clearly a need for more research. In addition, no study that was reviewed
investigated the combined effects of maternal diet, maternal exercise andlor
litter size on lactation performance as measured by mammary gland growth and
development and milk composition and yield.
IV. STATEMENT OF PURPOSE
Since glucose is considered the principle precursor for milk lactose and
de novo synthesis of milk fat (Williamson, 1980) it is conceivable that
cornpetition for glucose exists between exercising muscle and the mammary
gland. To this end, few studies have addressed the concept of maternal
exercise and altered glucose supply to the mammary gland during lactation.
There are no published studies which have examined the combined effects of
maternal carbohydrate restriction, exercise and litter size on lactation.
Therefore, the purpose of the this study was to investigate the cornbined effects
of materna1 dietary glucose restriction, chronic exercise and litter size on
lactation performance, specifically mammary gland, milk composition and
neonatal growth in rats. The hypothesis was that dietary glucose restriction,
chronic exercise and litter size would independently or in combination, modify
rat mammary gland composition, milk composition, and pup growth. Specific
objectives were to determine the independent effects of maternal dietary
glucose level (20%, 40%, 60% GLUC), activity level (exercise, sedentary) and
litter size (8 pups, 12 pups) as well as the interaction of dietary glucose level
and activity level on:
1) mammary gland composition by measuring mammary gland protein,
glycogen, lipid and DNA concentrations.
2) milk composition and milk yield by measuring milk protein, lactose and lipid
concentrations and 24 hr milk yield.
3) pup growth by measuring body weight.
CHAPTER 2 - MATERIALS AND METHODS
A. Experimental Design
A visual representation of the experimental design is shown in Figure
2.1. A 3*2*2 factorial design was used to investigate the effects of varying
levels of materna1 dietary glucose (20%, 40%, 60% GLUC), activity (chronic
exercise, sedentary) and litter size (8 pups, 12 pups) on mammary gland
composition and milk composition and milk yield. It should be noted here that
the term 'rnammary gland' refers to mammary tissue, not only glandular tissue.
The study was conducted in 2 phases. Pregnancy outcomes and early
neonatal survival rates were examined previously in phase 1 (Leccisi-Esrey,
Master's thesis, 1991). The combined effects of dietary glucose level, activity
level and litter size on lactation outcomes (biochemical mammary gland, milk
and plasma analyses and measurement of neonatal growth) were studied in
phase 2 and will be presented here. Table 2.1 shows the variables measured
in the pregnancy and lactation phases.
B. Animal Care and Experimental Protocol
One hundred thirteen (1 13) female Sprague-Dawley rats (Charles River
Canada Inc., St. Constant, Québec), weighing 175-200 g on arriva1 were used
for the experîment. All rats were individually housed in wire screen cages in a
temperature controlled room (21 + 2°C) with fluorescent lighting automated to
provide 12hr light daily (7h00-19h00) and were allowed to adjust to their
surroundings for 4-6 days. Standard Purina Rat Chow (Ralston Purina Co.,
Longueuil, Québec) and water were fed ad libitum until breeding. All rats
undenvent 18 days of exercise acclimatization on a motor driven treadmill and
were then divided into one of two activity groups: exercised (E) or sedentary (S).
Assignment to activity groups was based on running performance during the
FIGURE 2.1 EXPERIMENTAL DESIGN
EXERCISE ACCLlMATlZATlON 18 days
Sprague-Dawley rats
EXERCISED SEDENTARY
\
PREGNANCY (GdO) #
r PARTURITION-LACTATION (LdO) 1 \ #
LITTER f t *
MILKING & EXCISION OF MAMMARY GLANDS
TABLE 2.1
VARIABLES MEASURED IN THE PREGNANCY AND LACTATION PHASES OF THE STUDY
PREGNANCY LACTATION Cumulative materna1 feed intake Daiiy matemal food intake patterns Cumulative materna1 weight gain cumulative materna1 food intake Length of gestation Matemal weight patterns Number of implantation sites Maternal plasma: glucose Number of fetal resorptions insulin Number of pups born LDH Average birthweight of pups cortisol Neonatal pup mortality NEFA
Mammary gland: weight fat glycogen protein DNA cell site
Milk: protein fat lactose energy yield
Pup weight at Ld15 -
acclimatization period. Rats that did not run well during this period were
assigned to the sedentary group since they were unlikely to be able to complete
the exercise protocol during pregnancy. All rats were weighed individually to
the nearest gram every 1-2 days and followed the activity protocols throughout
pregnancy and lactation. All rats in the E group completed the exercise
protocol.
Pregnancy
Individual rats, weighing 245-255 g, were randomly placed with one of
13 Sprague-Dawley male breeders until impregnation. Presence of a vaginal
plug on the cage tray confirrned mating and the day was designated 'day O' of
gestation (GdO). The rats were then randomly assigned to one of three diet
groups (20, 40, 60% GLUC) within each activity group (n=15-20/subgroup).
These diets were provided ad libitum and were measured throughout
pregnancy and lactation. Diet intake was calculated every 1-2 days by
weighing the stainless steel feed cups on a Mettler electronic balance (Fischer
Canada, Montréal, QC.) to the nearest 0.1 g. Any spilled diet was weighed as
accurately as possible and was taken into account when calculating daily and
total diet intakes. On Gd1 9 al1 rats were transferred to plastic-bottomed
rnaternity cages with cedar chips for nesting and the day of parturition (Gd20-
21) was considered lactation day O (LdO).
Lacta tion
To test the effect of litter size on lactation outcomes and given that a
female rat usually has 12 mammae (Williams, 1976), a litter size of 12 pups was
chosen in order that al1 the dam's teats would potentially be occupied. A litter
size of 8 and not less was chosen to promote lactation and to prevent obese
pups since a model for lactation and not obesity was desired. Reducing litter
size has historically been a method for creating obese rat pups (Dugail et al.,
1986; Mela et al., 1987). Models for overfeeding have been created with M
pups/litter and underfeeding created with 216 pupsllitter (Knittle and Hirsch,
1968; Faust et al., 1980). Litter sizes between these 2 extremes were therefore
chosen for the study. Litters of 2 12 pups were generally culled to 12 and Mers
< 12 pups culled to 8 since among diet groups and between exercise groups no
significant differences were found for average number of implantation sites (1 5-
16), average number of fetal resorptions (1-2) and average number of pups
born to dams (Leccisi-Esrey, 1991). Culling took place on Ld3 to allow nursing
to establish. No pups were cross-fostered. Beginning in the morning of Ld12,
milk yields were determined by a modified version of the test-weighing
procedure used by Treadway and Lederman (1986). The period of pup
separation from the dams was reduced from 5 hours to 4 hours as separation
>5 hours has been shown to decrease milk production (Reddy and Donker,
1965). Pups were separated from their mothers for 4 hours, individually
weighed, allowed to suckle for 1 hour and then pups were weighed individually
again. This procedure was repeated 4 more times (total 25 hours) and the pup
weight differences from the 5 suckling periods were pooled for each litter to
represent 25 hour milk yield for each dam. Pooled weight differences were
divided by 25 hours and rnultiplied by 24 hours to obtain 24 hour milk yield.
Collection of milk samples for composition analysis was done on Ld15.
At least 12 hours post exercise for the E dams, rats were separated from their
Mers and within 2 hours were anesthetized by injection with Ketamine HCI
(CDMV Drugs, Montréal, QC.) at a dose of 30 mgkg body weight and were then
given an intraperitoneal injection of oxytocin (2 IU; PVU Inc., Victoriaville, QC.)
to stimulate milk flow. Within 20 minutes, milk was collected by hand stripping
of teats into Pasteur pipettes attached to a manual vacuum pump until no more
milk was obtained. Samples were stored at -80°C until analysis.
Between 10:OO and 14:OO rats were given another injection of Ketamine
HCI, blood was withdrawn by cardiac puncture and then al1 mammary gland
tissue was excised. Blood samples were centrifuged and the plasma stored at
-80°C All tissues were weighed, frozen in liquid nitrogen and stored at -80°C
until analysis. Guidelines were followed as outlined by the local animal care
cornmittee of McGill University and by the Canadian Council on Animal Care
(1984). The entire protocol was repeated 3 times with similar numbers of rats.
C. Exercise Protocol
A multi-lane motor driven treadmill was used for acclimatization and
exercising of rats. The treadmill was equipped with an electric shock grid;
however, it was not used since it would have introduced stress in addition to the
exercise that would not be present in the S animals. An observer was present
during al1 exercise sessions and rats were prodded from their hindquarters to
keep them running. Mild shock was used on the first set of rats for the first and
second days of the exercise acclimatization period; however, no rats were
shocked during gestation or lactation.
To keep environmental conditions as uniform as possible between
groups, S rats were placed in the same room as the E rats while they were
running. In addition, food and water were removed from the cages of the S rats
to keep access time to these constant for both activity groups.
Acclima tria tion
Following the 4-6 day adjustment period, al1 rats were acclimatized to an
exercise regime for 18 days which was considered the shortest time required for
the rats to become accustomed to running on a treadmill (Mottola et al., 1983).
Exercise acclimatization consisted of running on a motor-driven treadmill
initially at 10 rn/min.*5 min./day (-1.6 W of energyld) and gradually increasing
running speed and duration to 20 mlmin.*45 min.lday (-37.5 Wld) at the end of
the 18 day period. Watts of energy expended through exercise were calculated
with the following formula: work in kgbm = [(% grade) x (mlmin) x (durationmin) x
(body masskg)], with 6 kgem = 1 W (Cobrin and Koski, 1995). The level of
exercise at the end of the acclimatization period corresponds to approximately
70% VOzmax in non-pregnant rats (female rats-Brooks and White, 1978; male
and female rats-Bedford et al., 1979; male rats-Shepherd and Gollnick, 1976)
and is considered to be of moderate intensity. Similar exercise acclimatization
regimes with higher final intensities (Le. strenuous exercise at 280% V0Zmax)
have been used by others (Mottola et al., 1986 and 1989) for use with pregnant
rats. Live pups which would exhibit normal nursing behaviour were required to
investigate lactation outcornes. Rats were therefore exercised at moderate
intensity since high intensity exercise (80-88% V02rnax) has been shown to
increase postnatal pup mortality (Wilson and Gisolfi, 1980). In addition,
moderate intensity exercise was used to ensure that most of the pregnant rats
completed the exercise protocol. Attrition rates of 20% have been reported in
studies exercising pregnant rats at high intensity (i.e. 280% VOpmax - Mottola et
al., 1986 and Mottola et al., 1989).
Pregnancy
All E rats were exercised at 20 m/min.'45 min./day (-37.5 Wld) until
breeding. On Gd0 al1 E rats did not un , but resumed on Gdl. All rats were
running 20 m/min.*60 min./daye7 daydweek (-50 W/d) by Gd3 and continued
this routine until Gd20 (-77 W/d).
Lactation
Dams in the E group continued the exercise pmtocol at an intensity of 20
m/min.*45 min.ld (-44 W/d) on lactation day 3 (Ld3) and increased to 20
m/min.*60 min./day*7 daystweek on Ld5 (-60 Wtd) to Ld14 (-59 Wtd). Dams
did not run during Ld12 when milk yields were measured.
D. Experimental Diets
The experirnental and control diets (Table 2.2) were adapted from
experiments conducted by Fergusson and Koski (1 990). Rats consumed diets
and water ad libitum throughout pregnancy and lactation. Diets were
isoenergetic and provided approximately 4.15 kcal of metabolizable energy per
gram of dry weight. The 20% and 40% carbohydrate diets were formulated by
replacing carbohydrate energy from the 60% diet with equivalent energy from
soybean oil. The diet weight was made up with cellulose. All other elernents of
the diets remained constant (protein, vitamins and minerals) and were provided
in quantities that met NRC daily requirements for pregnant or lactating rats,
whichever was higher (NRC, 1978).
Considering that lactation outcomes were to be investigated and live
pups were required, the dietary glucose restrictions imposed were not severe.
Severe dietary carbohydrate restriction has been shown to result in abnormal
lactational petformance with ~ 6 % GLUC diets (Koski et al., 1990), reduced pup
growth with 112% GLUC diets (Koski and Hill, 1986; Koski et al., 1990) and
increased mortality of pups nursed by dams fed these restricted diets (Koski and
Hill, 1986, Koski et al., I W O ; Koski and Hill, 1990). Diets with 12% and 24%
GLUC have been shown to produce similar reproductive outcomes (number of
implantation sites, rates of in-utero death , resorption and live pups), pup
weights at Ld15 and materna1 body weights from Ld3-15 as a control diet of
60% GLUC (Lanoue and Koski, 1994). Maternal and pup growth were
therefore not compromised, but dams and pups may have been metabolically
compromised. Adding the stress of exercise to this model of potentially
TABLE 2.2
COMPOSITION OF CONTROL AND GLUCOSE (GLUC) RESTRICTED DIETSI
INGREDIENT CONTROL 40% GLUC 20% GLUC
Glucose* Soybean 013 Cellulose4 Casein5 Vitamin mixture6 Mineral mixture6 Methionine Sodium bicarbonate Metabolitable Energy - kcallg -kJ/g
Dry weight basis (grarns) *~extrose (anhydrous). ICN Biochernicals Canada Ltd., Montréal, Québec 30egummed soybean oil, Canada Packers Inc., Montréal, Quebec 4~lphacel. ICN Biochemicals Canada Ltd., MontrBal. Quebec 5~igh-nitrogen casein, ICN Biochemicals Ltd., Montréal, Quebec. Contains 90% protein (N16.25) %amin and mineral mixture composition as previously reported (Fergusson and Koski, 1990)
comprornised status would allow the dams' metabolisrn to be further
investigated, therefore a level of 20% GLUC was chosen for this experiment.
The 40% GLUC diet was chosen as an intermediate level of carbohydrate
between the 20% GLUC diet and the 60% GLUC control diet.
By using isoenergetic composite diets and varying the level of
carbohydrate, changing the level of fat cannot be avoided. Given the higher
energy density of dietary fat over carbohydrate (-38 kJ/g vs. -17 kJ/g), a smaller
amount (grams) of dietary fat than carbohydrate is required to replace the same
amount of calories. To illustrate, in the diets used for this study, there is a 40 g
difference in dietary GLUC between the control diet of 60% GLUC and the 20%
GLUC diet, but only a 15 g difference in fat for the same amount of energy.
Alterations in reproduction and lactation (Koski and Hill, 1986, Koski et al.,
1990; Koski and Hill, 1990) have resulted from materna1 diets providing a low
level of dietary carbohydrate of 0%-12% GLUC and thus a fairly high level of
dietary fat. In addition, milk fat was decreased with 12 and 24% GLUC diets
(Lanoue and Koski, 1994). It cannot be concluded that these alterations are
strictly carbohydrate driven, but for dietary fat to be responsible, rat metabolism
would have to be highly sensitive to any relatively small changes in dietary fat
compared to carbohydrate.
E. Quantitative Assays
i. Mammary Gland Composition
Mammary gland tissues were analyzed for total protein, glycogen, fat and
DNA concentrations.
Protein
For preparation of mammary gland tissue for the protein assay, a portion
of each mammary gland was homogenized in potassium phosphate buffer (-lg
tissue/lO mL buffer) using a Brinkmann Polytron PT-3000. Total protein
concentration of mammary gland tissue was determined using a modified
Hartree (1972) method. Prior to the assay, tissues were prepared as follows:
100 pL of homogenized sample were incubated with 400 pL of 0.1 N NaOH for
30 minutes at 37OC. The sample was centrifuged for 15 minutes at 4OC at 2500
rpm (-1300 g) and then 50 pL of the supernatant was transferred to a clean
tube with 950 pL phosphate buffer. Samples were not diluted to 1 mL with
water prior to the assay as indicated in Hartree (1972) (i.e. undiluted samples
were assayed). Assay reagent amounts as described in the original method of
Hartree (1 972) were doubled (i.e. 1.8 mL solution A, 200 pL solution B and 6
mL solution C were used). Bovine serum albumin (BSA) was used as the
standard. Results were expressed as mg/g wet tissue.
DNA
DNA was quantified using the method of Burton (1956). Prior to the
assay, samples were prepared by adding 1 mL-10% TCA to 1 mL sample
homogenate (as described above) and then adding 5 mL of 5% TCA. Samples
were centrifuged for 10 minutes at 2500 rpm (-1300 g). Pellets were
resuspended in 5 ml-5% TCA, covered and then heated in an 100°C waterbath
for 60 minutes. After centrifugation for 10 minutes at 2500 rprn (-1 300 g), 1 mL
of the supernatant was used for the assay. Calf thymus DNA diluted to 1 mg/rnL
water was used as the standard. Standard sets were made using 2.5% TCA
(trichloroacetic acid). Results were expressed as mglg wet tissue.
Glycogen
Mammary gland glycogen concentration was assayed using a rnodified
method of Lo et al. (1970). Weighed Kimax tubes with caps were cooled in dry
ice. Frozen, powdered tissue was added along with 800 PL-30% saturated
KOH. Tissues were allowed to thaw and were re-weighed. Six hundred jd- of
30% saturated KOH were added to each sample and the mixture gently
vortexed. Tubes were boiled in a waterbath for 45 minutes until a
hornogeneous solution was obtained. Tubes were cooled on ice for 10 minutes
and then 2 mL of 95% ethanol were added. Tubes were vortexed and cooled
on ice for 30 minutes and then centrifuged for 1 hour at -5°C. Resulting pellets
were dissolved in 1 mL de-ionized water. The assay was completed as per Lo
et al. (1970) except 6% phenol (31.5 mL of 88% phenol made up to 500 m l )
was used instead of 596 phenol. Results were expressed as mgig wet tissue.
Lipid
Total lipid contents of freeze-dried mammary glands were determined
gravimetrically after grinding and extraction with chlorofom-methanol using the
method from the Soxtec System HT2 (Tecator, Hoganas, Sweden). Results
were expressed as percentage of dry and wet weights.
ii. Milk Composition
Total milk protein concentration was determined as described above
except that 50 pL of raw milk was first incubated at 37OC in 450 pL of 0.1 N
NaOH. Lactose concentration was detennined by the orcinol-sulfate reaction
described by Svennerholm (1956). Total lipids were determined by a
colorimetric sulfuric acid-vanillin reaction kit (Boehringer Mannheim Canada,
Laval, QC, Cat. No. 124 303). All results were expressed as mg/mL milk.
iii. Plasma
Plasma glucose concentration was quantified by hexokinase
determination (Sigma Diagnostics, St. Louis, MO, Cat. No. 16-20) using an
Abbott VP Super System (Irving, TX). Plasma insulin was measured using an
1251 insuline (sic) RIA coated tube kit (Immunocorp, Montréal, QC, Cat. No. Ktsp-
11 002). Plasma lactose dehydrogenase activity (LDH) was measured using a-
gent@ LDH Clinical Chemistry Reagent (Abbott Laboratories Diagnostics
Division, Abbott Park, IL, Cat. No. 6012-02) and an Abbott VP Super System
(1 rving , TX). Plasma cortisol was determined using an IrnmunoChernTM Cortisol
1251 kit (ICN Biochemicals, Inc., Costa Mesa, CA, Cat. No. 07-221 102). Plasma
non-esterified fatty acids were measured with a NEFA C kit (Wako Chemicals,
Richmond, VA, Cat. No. 994-75409E) modified to a microprocedure (al1
quantities of sarnple and reagent were reduced to 115 of the amounts normally
used) .
F. Data Management and Statistical Analyses
The experimental design was a 3*2*2 factorial with 3 levels of dietary
glucose (20%, 40%, 60%), 2 levels of exercise (chronic exercise, sedentary)
and 2 litter sizes (8, 12 pups). Data management was carried out using Lotus
v.3.1 (1989) and IBM Personal Editor v.2. All,analysis was carried out using the
SAS System for Windows v.6.10 (1994, Cary, NC).
Data was tested for homogeneity of variances using Bartlett's test (Steel
and Torrie, 1980). Data sets with non-homogeneous variances were log
transformed (MG fat dry weight, plasma cortisol, dam pregnancy weight pattern
and average daily milk energylpup) or, when this transformation did not resuit in
homogeneous variances, observations were weighted by the reciprocal of the
group variance for each of the following groups: total food intake at the end of
lactation, MG wet weight, MG weight as % body weight, MG fat wet weight, milk
protein, plasma insulin and individual pup weight at Ld15. Variances of 'day'
groups for daily pregnancy and lactation food intake patterns of dams, as well
as 2-day pregnancy weight gain pattern were found to be non-homogeneous
and were not made homogeneous by log transformation. Given that only effects
within the same day and not from day to day were to be compared non-
weighted data were used for analysis.
All data except pup weights and food intake and weight patterns were
then analyzed (see Appendix 1 for SAS model statements) by analysis of
variance (ANOVA) using the general linear models procedure (proc glm). All
data except, pup weights, plasma parameters and food intake and weight
patterns were analyzed by analysis of covariance (ANCOVA) also using the
general linear models procedure (proc glm) with dam pre-pregnancy weight
(GdO) as a covariate. Daily dietary intake and dam weight patterns were
analyzed by repeated measures ANOVA using the mixed procedure (proc
mixed) which allows more appropriate analysis of models with both randorn and
fixed effects. Diet, exercise, litter size and day were considered fixed effects
since we were interested in the differences between the levels within each
effect group (i.e. were there any significant effects of diet, exercise, litter size or
day?). Dam was considered a random effect since we were not interested in
differences between individual dams, but in the variability in the population of
dams. Individual pup weights at Ld15 were analyzed by a nested ANOVA and
average pup weightldam was analyzed by a nested ANCOVA separately using
milk (yield, protein, fat, lactose, total kcaVmL and total kcallday) or diet intake
parameters (total diet intake during pregnancy and lactation) as covariates.
60th analyses were done using the mixed procedure again because of more
appropriate handling of the fixed (diet, exercise, litter size) and random (dam)
effects. Significant effects were analyzed using Bonferroni's test for multiple
comparisons.
Linear regression and correlation analysis was carried out on the milk
parameters using the regression procedure (proc reg). In addition, multiple
regression was done to determine predictors of milk yield, milk protein, milk fat
and milk lactose, as well as, average pup weight at Ld15.
CHAPTER 3 - RESULTS
Please note that al1 result tables and figures can be found at the end of
chapter 3 on pages 58 to 93. For the sake of clarity, error bars were not
included in the line graphs of the repeated measures data.
A. ~aternal Food lntake
Pregnancy
Cumulative (total) maternal food intake during gestation (Gd0 to birth) is
shown in Table 3.1 and Figure 3.1. At the end of gestation there were no
significant main effects of diet or exercise and no significant interaction of
diet*exercise on cumulative maternal food intake. However, when pre-
pregnancy weight (weight at GdO) was used as a covariate in the analysis, the
main effect of diet was found to be significant (F=3.65; p=0.0301). Dams fed the
20% GLUC diet consumed significantly less food than dams fed the 60% GLUC
diet. Total food consumed by dams fed the 40% GLUC diet was not significantly
different from that of the 20% GLUC or 60% GLUC diet groups. The relevance
of this result, considering that prepregnancy weights were not different by diet,
exercise or diet*exercise groups is not clear.
Repeated measures ANOVA of daily food intakes throughout pregnancy
(Gd0-21) are shown in Table 3.2 and Figures 3.2a and b. Significant
interactions were found between day of pregnancy and diet and day of
pregnancy and exercise indicating that the effect of day was not the same within
each diet and exercise effect groups. There was no significant interaction
between day of pregnancy and diet'exercise groups.
Lactation
Cumulative (total) maternal food intake during lactation (birth to Ld14) is
also shown in Table 3.1 and Figure 3.1. At the end of lactation, there were no
statistically significant main or interaction effects of diet, exercise or
diet*exercise on cumulative food intake. However, there was a significant main
effect of litter size. Dams that nursed larger litters (12 pups) consumed
significantly more food than dams that nursed smaller litters (8 pups).
Repeated measures ANOVA of daily food intakes throughout lactation
(Ld0-14) are shown in Table 3.2 and Figure 3.2a and c. Significant interacti~ns
were found between day of lactation and diet and day of lactation and litter size.
Given the significance of the day of lactation and litter size interaction, the
significance of the main effect of litter size is not useful as it is unknown if the
significance of litter size is due to litter size effects alone, the interaction
between litter size and day or both. There were no significant interactions
between day of lactation and exercise or day of lactation and diet'exercise
groups indicating that the effect of day was the same within each effect group
du ring lactation.
B. Matetnal Body Weight
Pregnancy
Table 3.3 and Figure 3.3 show final maternal weight at the end of
pregnancy (prior to parturition). There were no significant main or interaction
effects of diet or diet'exercise on maternal weight at the end of pregnancy;
however, there was a significant main effect of exercise. Chronically exercised
dams weighed less at the end of pregnancy compared to sedentary dams.
When pre-pregnancy weight was used as a covariate in the analysis, the main
effect of diet was significant (F=3.40; p=0.0386), such that body weights of
dams fed the 20% GLUC diet were significantly lower than those of dams fed
the 60% GLUC diet at the end of pregnancy. The body weights of dams fed the
40% GLUC diet were not significantly different from those of dams fed the 20%
GLUC or 60% GLUC diets. As with cumulative materna1 food intake at the end
of pregnancy, the relevance of this result is not obvious.
Table 3.4 and Figure 3.4a, b, cl d and e show results from repeated
measures ANOVA of pregnancy weights and weight gains of dams taken every
other day. A significant interaction effect between diet and exercise was found
for average pregnancy weight with sedentary activity resulting in heavier body
weights when dams were fed the 40% GLUC diet compared to the 20% GLUC
diet. There were no significant interactions found between day and diet, day
and exercise or day and diet*exercise groups for pregnancy weight. Significant
interaction effects between day of pregnancy and diet and day of pregnancy
and exercise were found for average 2-day weight gain during pregnancy. This
indicates that the effect of day was not the same within each diet group and
exercise group. Significant differences are indicated with asterisks on Figure
3.4e.
Lactation
Final matemal weight at the end of lactation (Ld15) is shown in Table 3.3
and Figure 3.3. No significant main or interaction effects of diet, exercise, litter
size or dieteexercise were found for final matemal weight at the end of lactation.
Repeated measures ANOVA of lactation weight taken every other day is
shown in Table 3.4 and Figure 3.4a and f. The main effect of diet was
significant for average dam weight across the period of lactation. The average
weight across the period of lactation was significantly lower for dams fed the
20% GLUC diet than those of dams fed the 40% and 60% GLUC diets. The
main effects of exercise and litter size were not significant. No interactions
between day and diet, exercise, litter size or diet*exercise were found.
C. Plasma Analyses
Analyses of maternai plasma parameters are shown in Table 3.5 and
Figures 3.5a, b and c. It should be noted here that since blood samples were
not taken immediately post-exercise, any significant effect attributed to exercise
should be interpreted as a net effect of chronic exercise and not a direct effect
of a single bout of exercise.
Glucose
Analysis of lactation day 15 (Ld15) plasma of dams revealed that size of
litter affected plasma glucose levels. Dams that nursed larger Mers (12 pups)
had significantly lower plasma glucose levels than dams that nursed smaller
litters (8 pups). No statistically significant main effects of diet and exercise nor
an interaction effect between the two were found.
Insulin
Significant diet and exercise effects on plasma insulin levels were found.
Dams fed the 20% GLUC diet had significantly higher plasma insulin levels
compared to dams fed the 60% GLUC diet. lnsulin levels of dams fed the 40%
GLUC diet were not significantly different from those of both the 20% GLUC and
the 60% GLUC diets. Chronically exercised dams had significantly lower
plasma insulin levels compared to sedentary dams. No significant main effect
of litter size, nor an interaction between diet and exercise were found.
Lactate Dehydrogenase (LDH)
Plasma LDH activity was also significantly affected by diet, as dams fed
the 20% GLUC diet produced higher LDH activity compared to the 40% and
60% GLUC diets. No other significant main effects of exercise or litter size nor
an interaction between diet and exercise were found.
Non-esterified Fatty Acids (NEFA)
No significant main or interaction effects of diet and exercise on plasma
NEFA levels were found; however, dams that nursed a litter size of 12 pups had
significantly increased plasma NEFA levels.
Cortisol
Analysis of plasma cortisol levels did not reveal any significant main
effects of diet, exercise and litter size, nor a significant interaction between diet
and exercise.
D. Mammary Gland Composition
The effects of dietary glucose level, chronic exercise and litter size on
Ld15 mammary gland composition are shown in Table 3.6. and Figures 3.6a
and b.
Mamrnary Gland Weight
A significant main effect of litter size was found on mammary gland wet
weight and mammary gland wet weight expressed as a percentage of body
weight. Marnmary glands of dams that nursed 8 pups weighed less and
represented a smaller percentage of body weight than those that nursed 12
pups. No other significant main effects of diet, exercise or interactions between
diet and exercise were found.
Fat
C hronic exercise was found to signif icantly decrease mammary gland fat
content (percent wet and dry weight) compared to sedentary activity, No
significant effects of diet or litter size or interactions between diet and exercise
were found.
Protein, Glycogen, DNA, Ce11 Sire
Mammary gland protein concentration was affected by litter size with
mammary glands from dams that nursed larger litters having higher protein
concentrations. Mammary protein concentration was not significantly affected
by diet, exercise or the interaction between diet and exercise. In addition, no
significant main or interaction effects of diet, exercise, litter size or diet'exercise
were found on mammary gland glycogen, DNA or cell size as represented by
PR0TElN:DNA.
E. Milk Composition
The effects of dietary glucose level, exercise training and litter size on
Ld15 milk composition, energy and yield are shown in Table 3.7 and Figures
3.7a, b, c and d.
Protein
Milk protein concentration was altered by the level of dietary glucose.
Specifically, dams that consumed a 20% glucose diet had significantly higher
milk protein than dams that consumed 40% or 60% glucose diets. Exercise,
litter size or the interaction between diet and exercise did not significantly affect
milk protein concentration.
Fat
Diet and chronic exercise were found to affect milk fat concentration. In
contrast to the findings with milk protein, as dietary glucose increased, the
concentration of milk fat increased significantly. Fat was also significantly
increased in milk from dams which were chronically exercised compared to
dams which were sedentary. No significant litter size and interaction effects
were found for milk fat concentration.
Lactose
Litter size did not significantly affect milk lactose; however, a significant
interaction between diet and exercise was found for milk lactose concentration
(see Figure 3.7b.). The difference between milk lactose concentrations of
chronically exercised and sedentary dams fed the 20% GLUC diet was
significantly greater than the difference between milk lactose concentrations of
chronically exercised and sedentary dams on 40% and 60% GLUC diets. In
other words, chronic exercise in rats fed the 20% GLUC diet resulted in a
significantly lower milk lactose level compared to sedentary activity in rats on
the same diet, whereas no such difference was found in the 40% GLUC and
60% GLUC diet groups.
Energy
The level of dietary glucose altered milk energy (kcaVmL and kcallday)
as dams fed 20% or 40% GLUC diets had milk with significantly less energy per
mL and per day than dams fed a 60% glucose diet. Chronic exercise, litter size
and the interaction between diet and exercise did not significantly affect milk
energy.
Litter size altered 24 hour milk yield. Dams that nursed larger litters (12
pups) produced significantly more milk compared to dams that nursed srnaller
litters (8 pups). The other main and interaction effects of diet, exercise and
diet*exercise were not significant.
F. Pup Analyses
Analyses of pup results are show in Table 3.8 and Figures 3.8a and b.
Analysis of individual pup weights on Ld15 revealed significant main
effects of diet and litter size. Dams that consumed the 40% GLUC diet had pups
that weighed significantly more on Ld15 than dams that consumed the 60%
GLUC diet. Dams that consumed the 20% GLUC diet had pups that did not
differ significantly in weight (Ld15) from those from dams fed the 40% GLUC
and 60% GLUC diets. Dams that nursed larger Mers had pups that weighed
significantly less on Ld15 than pups from dams that nursed smaller litters.
When average pup weight (Ld15) for each dam was analyzed using 24
hour milk yield, milk fat concentration and total calorieslrnL as separate
covariates, the main effect of diet was no longer significant (milk yield: F= 2.69,
p=0.0759; milk fat: Fs2.20, p=O.ll76; kcallmL: F=2.31, p=O.lO62).
Significant main effects of diet and litter size were found on average daily
milk volumelpup. Pups nursed by dams fed the 20% GLUC diet received
significantly less milklday compared to pups nursed by dams fed the 40% or
60% GLUC diets. In addition, pups nursed in larger litters of 12 received
significantly less milktday than pups nursed in litters of 8. There was no
significant effect of exercise nor a significant interaction between diet and
exercise on average daily milk volume/pup.
Average daily milk energylpup was also significantly affected by diet and
litter size. Diet had a graded effect on milk energy/pup as pups received
significantly increasing amounts of milk energylday as glucose increased in the
diets of their mothers. Pups nursed in larger litters received significantly less
milk energylday than those nursed in smaller litters. As with milk volumelpup,
there was no significant main effect of exercise nor interaction between diet and
exe rcise.
Average daily milk faüpup followed a similar pattern to milk energylpup.
Pups received significantly increasing amounts of milk fat/day as glucose
increased in the diets of their nursing dams. In addition, pups nursed in larger
litters received significantly less milk fat/day than pups nursed in smaller litters.
There was also no significant main effect of exercise and no interaction
between diet and exercise on daily milk faüpup.
G. Correlation and Regression Analyses
Milk Correla tion and Regression Analyses
Correlation analysis showed that milk lactose concentration (mglml) was
not significantly associated with milk protein or fat concentrations (mg1mL;
Pearson correlation coefficient = 0.139, p = 0.195 and 0.078, p = 0.455
respectively). However, total milk lactose content (9124 hours) was significantly
and positively correlated with total milk protein and fat contents (gl24 hours;
lactose vs. protein, Pearson correlation coefficient = 0.682, p = 0.0001 and
lactose vs. fat, 0.465 , p = 0.0001). Regression of total milk lactose content on
24 hour milk yield revealed that 59% of the variability of milk yield could be
explained by milk lactose (F = 97.607, p = 0.0001).
Regression Analysis - Preciictors of Milk Composition and Yield
Multiple regression analysis of diet, exercise and litter size, MG weight as
a percentage of body weight, MG fat wet weight, MG protein, MG glycogen, MG
cell size, total diet intake during pregnancy and total diet intake during lactation
on milk composition and yield was carried out. Milk fat was predicted by level of
dietary glucose. As dietary glucose increased from 20% to 40% to 60%, milk fat
concentration increased by 38.45 + 7.62 mghL (p = 0.0001). Milk protein was
predicted by dietary glucose level and total diet intake during pregnancy. In
contrast to milk fat, as dietary glucose increased from 20% to 6074, milk protein
decreased by 7.56 f 2.12 mg/rnL (p = 0.0007) and for every gram of diet intake
during pregnancy, milk protein decreased by 0.06 t 0.03 mg/mL (p = 0.0389).
Milk lactose concentration was not predicted by any variable used in the
regression analysis. Milk yield was predicted by MG fat wet weight and total
diet intake (g) during lactation. For every 1 percent increase in mammary gland
fat and 1 gram increase in diet (-17 kJ) intake during lactation, milk yield
decreased by 0.46 rt 0.20 mL (p = 0.0249) and increased by 0.03 k 0.01 mL (p =
0.0430) respectively.
Regression Analysis - Predictors of Pup Weight
Multiple regression analysis of milk protein, fat, lactose and yield per pup
on the average pup weight (Ld15) for each dam revealed that milk yield per pup
was predictive of average pup weight. At Ld15, for every 1 mL increase in milk
yield per pup the average pup weight increased by 3.47 + 0.62 g (p = 0.0001).
TABLE 3.1 Cumulative materna1 food intake (g) at the end of pregnancy and lactatlonl
EFFECT DlET 20% GLUC
40% GLUC 465.22 f 9.94 508.17 % 8.52 (34) (34)
60% GLUC 462.15 f 10.26 485.94 * 12.1 4 (31) (31 )
EXERCISED (E) 451.67 & 8.07 51 1.64 + 10.53 (50)
SEDENTARY (S) 456.43 f 8.33 (50)
492.57 t 7.81 (47) (47)
LITTER SlZE 8 PUPS
12 PUPS
STATISTICAL ANALYSIS EFFECT:
DlET
PREGNANCY LACTATION F / D values F / D values
2.85 / 0.0631 1.51 / 0.2266 EXERCiSE 0.1 9 / 0.6652 2.52 / 0.1 156 DIETEXERCISE 0.32 / 0.7263 0.1 1 / 0.8934 LllTER SlZE - 30.43 / 0.0001
1. End of pregnancy includes parturition. End of lactation = lactation day 14 (Ldl4). 2. Values are least square means (SM) f SELSM with number of dams in parentheses. 3. Values are weigMed l e ~ t square means f SELSM with number of dams in parentheses. LSM within a group not sharing the same superscript are significantly diierent from one another. pe0.05. 4. D20/40/60 = 20%/40%/60% GLUC diets. E = chronically exercised. S = sedentary.
FIGURE 3.1 The effect of a) dietary glucose level, b) chronic exercise and c) litter sire on cumulative materna1 food intake (g) at the end of pregnancy and lactation
1. Values are least square means (LSM), End of pregnancy includes parturition. End of lactation = lactation &y 14 (Ld14). Colurnns within a lactation or pregnancy group not sharing the same letter are significantiy different from one another, ~ 0 . 0 5 .
TABLE 3.2 Repeated measures ANOVA of daily food intake during pregnancy
of dietary glucose level, chronic exercise, and lactation: effects litter size and dayl
PREGNANCY 0 - - EFFECT
DlET - 20% GLUC
40% GLUC
60% GLUC
EXERCISE EXERCISED (E)
SEDENTARY (S)
LITTER SlZE 8 PUPS
12 PUPS
STATISTICAL ANALYSE EFFECT: F / o values
2.52 / 0.0804 0.04 / 0.8502 0.28 / 0.7595 - 10.55 / 0.0001 2.51 / 0.0001 3.06 / 0.0001 0.58 / 0.9866 -
F / p values
DlET EXERCISE DIFIYEXERCISE LllTER SlZE DAY DIWDAY EXERCISE*DAY DIWEXERCISE*DAY LITTER SIZE*DAY
1. Values are least square means (LSM) f: SELSM. LSM within a group not sharing the same superscript are significantly different from one another, pc0.05. Day effects indicate within subject variation. See text for further explmation. 2. D20/40/60 = 20%/40%/60% GLUC diets. E = chronically exercised. S = sedentary.
FIGURE 3,2a The effect of a) dietary glucose level, b) chronic exercise and c) litter size on daily materna1 food intake during pregnancy and lactation1
1. Values are least square means (LSM). In graph 'c', since there was a signifiant interaction between litter size and day. the ciifference between litter sizes is not shown. See text for further expianation.
FIGURE 3.2b The effect of a) dietary glucose level and b) chronic exercise on materna1 food intake pattern during pregnancyl
I -20% GLUC -40% GLUC
1. Values are least square means (LSM). See Table 3.2 - DIWDAY and EXERCISE*DAY for significance, p < 0.05.
FIGURE 3.2~ The effect of a) dietary glucose level, b) chronic exercise and c) litter size on materna1 food intake pattern during lactation1
-40% GLU
1. Values are least square means (MM). See Table 32 - DIETDAY, EXERCISE*DAY and LllTER SIZE*DAY for significance, p c 0.05. For graph 'c', within ail days except day O, food intake of dams that nursed 8 pups is significantiy different from that of dams that nursed 12 pups.
TABLE 3.3 Final maternal weight (g) at the end of pregnancy (prior to parturition) and at the end of lactation (Ld15)I
EFFECT DlET - 20% GLUC 40% GLUC 60% GLUC
EXERCISE EXERCISED (E) SEDENTARY (S)
LITTER SlZE 8 PUPS 12 PUPS
STATISTICAL ANALYSE EFFECT: DIET EXERCISE DIET * EXERCISE LlTiER SlZE
PREGNANCY LACTATION
PREGNANCY
F / P values 1.76 / 0.1 780 5.44 / 0.0222 O. 1 8 1 0.8328
LACTATION
F / D values 1 .O8 / 0.3441 1.60 / 0.2102 0.1 1 / 0.8992 0.00 1 0.9593
1. Values are least square means (LSM) f SELSM with number of dams in parentheses. LSM within a group not sharing the same superscnpt are signifcantly different from one another, pc0.05.2. D20/40/60 = 20%/400/d60°/o GLUC diets. E = chronically exercised. S = sedentary.
FIGURE 3.3 The effect of dietary glucose level, chronic exercise and litter size on materna1 weight (g) at the end of a) pregnancy (prior to parturition) l and b) lactation (Ld15) l
Ii 20% GLUC ~140% GLUC
60% GLUC
EXERCISED SEDENTARY
B I DIET EXERCISE
EXERCISE LITTER SIZE
1. Values are least square means (LSM). Columns within a group not sharing the same letter are significantiy different from one another, ~ ~ 0 . 0 5 .
TABLE 3.4 Repeated measures ANOVA of dam welght during pregnancy and lactation and 2-day weight gain during lactation: effects of dietary glucose level, chronic exercise, litter size and dayl
PREGNANCY WElGHT (g12 PREGNANCY WElGHT GAIN LACTATION WElGHT (g13 EFFECT -.- (gl2 d a ~ ~ ) ~
20% GLUC
40% GLUC
60% GLUC
EXERClSE EXERCISED (E)
SEDENTARY (S)
LITTER SlZE 8 PUPS
12 PUPS
TABLE 3.4 CONTINUED,
STATISTICAL ANALYSE PREGNANCY WEIGHT PREGNANCY WEIGHT GAIN LACTATION WT EFFECT: F/ovailues ELUalUS F / D values
DlET EXERClSE D*E Ll l lER SlZE DAY DIETDAY EXERCISE'DAY DlET*EXERCISE*DAY UTTER SIZE'DAY
1. Least square means (LSM) within a group not sharing the same superscript are significantly different from one another, pc0.05. Day effects indicate within subject variation. See text for further explanation. 2. Values are back transformed LSM (analysis done on log transformed data). Since the diet'exercise interaction was significant, differences within the main effect groups of diet and exercise are not shown. See text for further explanation. 3. Values are LSM f SELSM. Since diet'day and exercise'day interactions were significant, differences within main effect groups of diet and exercise are not shown. See text for further explanation. 4. D20140160 = 20%/40%/60% GLUC diets. E = chronically exercised. S = sedentary.
FIGURE 3.4a The effect of a) dietary glucose level, b) chronic exercise and c) litter size on daily materna1 weight during pregnancy and lactation1
60% GLUC
E X E R C I S E D ~
1. Values are least square means (LSM). Columns within a lactation or pregnancy group not sharing the same lefier are significantly dierent frorn one another, p e 0.05. Since a significant interaction was found between diet and exercise for pregnancy weight, dierences within main effect groups of diet and exercise are not shown. See text for further explanation.
FIGURE 3.4b The effect of dietary glucose level a and chronic exercise on materna1 2- day weight gain during pregnancyl
g 12 days
i20% GLUC ~ 4 0 % GLUC i60% GLUC
EXERCISED m SEDENTARY w
1. Values are least square means (LSM). Since significant interactions were found between diet and day and exercise and day, any differenceswithin main effect groups of diet and exercise are not shown. See text for further explanation.
FIGURE 3 . 4 ~ Interaction effect between dietary glucose and chronic exercise on daily materna1 weight during pregnancyl
O t I I I I I I
20% 40% 60% GLUC GLUC GLUC
1. Values are back transfomed lest square rneans (LSM). See text for significances, ~ 0 . 0 5 .
FIGURE 3.4d The effect of a) dietary glucose level and b) chronic exercise on materna1 weight pattern during pregnancyl
100 -- 1-40% GLUC
50 -- -60% GLUC
O 1 L I I 1 I 1 I I I
D ~ Y
O 2 4 6 8 10 12 14 16 18 20 21
1. Values are leaçt square means (LSM). See Table 3.4 - DIETDAY and EXERCISE'DAY for significance. p c 0.05.
FIGURE 3.4e The effect of a) dietary glucose level and b) chronic exercise on 2 day materna1 weight gain pattern during pregnancyl
1. Values are least square means (LSM). ' 20% GLUC diet significantly different f rom 40% and 60% GLUC diets OR exercised significanüy different from sedentary, ** al1 three diets significantly different from one another. ***20% and 40% GLUC diets significantly different from 60% GLUC diet. pc0.05.
FIGURE 3.4f The effect of a) dietary glucose level, b) chronic exercise and c) litter size on maternel weight pattern duting lactation1
1. Values are le& square means (LSM). See Table 3.4 - DIWDAY, WERCISE'DAY and LITTER SIZEeDAY for significance, p<0.05.
TABLE 3.5 The effecte of dietary glucose Ievel, chronic exercire and Iitter size on Ld15 dam
GLUCOSE INSULIN LACTATE CORTISOL NEFA
20% GLUC
40% GLUC
60% GLUC
EXERClSE EXERCJSED (E)
SEDENTARY (S)
ER SlZE 8 PUPS 12 PUPS
TABLE 3.5 CONTINUED.
STATlSTlCAL GLUCOSE INSULIN LACTATE CORTISOL NEFA ANALYSE DEHYDROGENASE EFFECT: E4Udlfm Eb2Yak EthYdM F / D values F / Q values Ol€T 0.58 / 0.561 1 3.63 / 0.0306 3.58 / 0.031 9 1.50 / 0.2294 0.75 / 0.4785 EXERCISE 1.77 / 0.1871 5.10 / 0.0264 3.75 / 0.0559 0.75 / 0.3893 0.31 / 0.5785 D * E 0.53 / 0.5918 0.01 / 0.9935 1.62 / 0.2033 2.82 / 0.0654 2.83 / 0.0678 Ll l lER SlZE 7.12 / 0.0091 0.03 1 0.8714 0.1 5 / 0.6962 2.00 / O. 161 2 5.57 / 0.021 9
1. Number of dams in parentheses. Least square means(LSM) within a group not sharing the same superscript are significantly different from one another, pc0.05. 2. Values are least square means (LSM) I SELSM. NEFA = non-esterified fatty acids. 3. Values are weighted LSM I SELSM. 4. Values are back transformed LSM (analysis done on log transforrned data). 5. 020/40/60 = 20%/40%/60% GLUC diets. E = chronically exercised. S = sedentary.
FIGURE 3.5a The effect of dietary glucose level, chronic exercise and litter size on Ld15 dam plasma a) glucose1 and b) insulin concentrations*.
DlET LITTER EXERCISE . SIZE
20% GLUC B 40% GLUC c 60% GLUC
a EXERCISED i SEDENTARY m m 8 PUPS pu 12 PUPS
DlET EXERCISE LIiTER SlZE
1. Values are least square means (LSM). Columns within a group not sharing the same letter are significantly different from one another, p4.05. 2. Values are weighted LSM. Colurnns within a group not shanng the same letter are significantly different from one another, p<0.05.
FIGURE 3.5b The effect of dietary glucose level, chronic exercise and litter sire on Ld15 dam plasma a) lactate dehydrogenasel and b) non-esterified fatty acids2.
1 20% GLUC
~140% GLUC 1160% GLUC I
EXERCISED i SEDENTARY H m 8 PUPS m l 2 PUPS
1. Values are least square means (LSM). 2. Values are weighted LSM. 1.2. Columns within a group not sharing the same letter are significantly different from one another, p<o.os.
FIGURE 3 . 5 ~ The effect of dietary glucose level, chronic exercise and litter sire on Ld15 dam plasma cortisol concentrationsl.
LlïïER SIZE
1. Values are back transformed least square means.
m20% GLUC ~ 4 0 % GLUC ~ 6 0 % GLUC m
EXERCISED i SEDENTARY
mi8 PUPS 1112 PUPS I
TABLE 3.6 The effect8 of dietary glucose level, chronic exercise and lltter size on Ld15 mammary gland composition1
FAT FAT GLYCOGEN PROTEIN DNA CELL SlZE
20% GLUC
40% GLUC
Ml% GLUC
EXERCISE EXERClSED (El SEDEMARY (SI
R x 5 D20"E
D20'S
D4O'E
DM*S
D60*E
D60"S
LllEmE 8 PUPS
' (49) 12 PUPS 21.57 f 0.32~
TABLE 3.6 CONTINUED.
STATlSTlCAL FAT FAT ANALYSIS WET Wï' % BODY WT (% dry wt) (% wet wt) GLYCOGEN PROTEIN DNA CELL SlZE MAIN FFECT: F/D values F / p v a l m F & i F m r , Y a i u e s F a F m F- F 1 I v a b s DlET 2.56 1 0.1000 2.34 1 0.1032 0.14 1 0.8678 0.57 1 0.5671 0.96 1 0.3885 1.85 10.1 633 0.33 1 0.7222 0.12 / 0.8836 EXERCISE 1.97 10.1642 1.51 10.2221 18.54 10.0001 17.47 10.0001 0.25 1 0.6151 0.03 10.6599 3.71 10.0571 2.58 10.1117 D * E 0.1910.8258 2.0010.1423 0.9510.3889 0.4210.6603 0.2510.7771 0.0910.9111 0.25/0.7823 0.0710.9367 LlllER SlZE 14.21 1 0.0003 8.20 / 0.0054 3.24 / 0.0750 1.77 / 0.1872 1.97 / 0.1644 4.90 1 0.0294 1.1 6 / 0.2841 0.24 1 0.6265
1. Number of dams in parentheses. Least square means (LSM) within a group not sharing the same superscript are significantly different from one another, pc0.05. 2. Values are weighted LSM i SELSM. 3. Values are back transformed LSM (analysis done on log transformed data). 4. Values are LSM fr SELSM. 5. D20140160 = 20%/40%/60% GLUC diets. E = chronically exercised. S = sedentary.
FIGURE 3.6a The effect of dietary glucose level, chronic exercise and litter size on Ld15 mammary gland content2 and c) glycogen content 3
a) weightl, b) fat
i 20% GLUC s 40% GLUC i 60% GLUC
EXERCISED i SEDENTARY
ri 8 PUPS RI12 PUPS
WET WEIGHt (g) % BODY WEIGHT
FAT (% dry wt) FAT (% wet wt)
-7 -
GLYCOGEN
1. Values are weighted lest square means (LW). 2. Percent dry weight fat vaiues are back transformed LSM. Percent wet weight fat valies are weigMed LSM. 3. Values ara LSM. 4. Unitç as indicated on x-a&. 1,2,3. Columns within a group not shanng the same letter are significantiy different from one another, pc0.05.
FIGURE 3.6b The effect of dietary glucose level, chronic exercise and litter size on Ld15 mammary gland a) protein contenti, b) DNA content and c) cell size 1.
r 20% GLUC ~ 4 0 % GLUC 1060% GLUC
EXERClSED m SEDENTARY
m8 PUPS a12 PUPS n
DNA
CELL SUE
1. Values are le& square means (LSM). Columns within a group rot sharing the sarne letter are significantiy different frorn one another. pc0.05.
TABLE 3.7a The effects of dietary glucose level, chronlc exercise and litter sire on Ldl5 milk composition, energy and yieldl
PROTEIN FAT UCTOSE ENERGY ENERGY YlELD
20% GLUC
40% GLUC
60% GLUC
EXERClSE EXERCISED (E)
SEDENTARY (S)
l.llmum 8 PUPS 12 PUPS
TABLE 3.7a CONTINUED.
STATISTICAL ENERGY ENERGY LmAwls PROTEIN FAT LACTOSE (kcalJmL) (kcallday) YlELD EFFECJ: - uEd4d= uMu= F / D values F/ovalues F 1 D values DJET 21 -79 / 0.0001 15.29 1 0.0001 0.89 1 0.41 62 14.13 / 0.0001 9.37 / 0.0003 2.28/ O.llOS EXERCJSE 0.32 10.5755 4.54 10.0358 0.86 10.3559 1.88 / 0.1739 0.23 / 0.6325 1.37 / 0.2471 0 * E 2.94 / 0.0594 0.83 / 0.4405 3.66 / 0.0298 1.38 / 0.2567 0.93 / 0.3994 O. 12 / 0.8859 Ll7TER SlZE 0.38 / 0.5372 1.52 1 0.2202 1.24 / 0.2693 1.18 / 0.2810 0.21 / 0.6474 16.12 / 0.0002
1. Number of dams are in parentheses. Least square means(LSM) within a group not sharing the same superscnpt are significantly different from one another, pe0.05. 2. Values are weighted LSM f SELSM . 3. Values are LSM f SELSM. 1 kcal = 4.18 kJ. 4. D20/40/60 = 20%/40%/60% GLUC diek E = chronically exercised. ES = sedentaty.
FIGURE 3.7a The effect of a) dietary glucose level, b) chronic exercise and c) litter size on Ld15 milk composition^.
i20% QLUC O 40% GLUC i 60% QLUC u
PROTElN FAT LACTOSE
PROTEIN FAT LACTOSE
0 8 PUPS
120
PROTEIN FAT LACTOSE
1. Values are least square means (LSM). Protein values are weighted LSM. Columns within a group not sharing the sarne letter are signlicantly dierent from one another, peO.05.
FIGURE 3.7b INTERACTION EFFECT BETWEEN DIETARY GLUCOSE AND CHRONIC EXERCISE ON MILK LACTOSE CONCENTRA~ION
20% GLUC 40% GLUC 60% GLUC
1. Values are least square means (LSM) f standard error of LSM. - See text for significance, p c 0.05.
FIGURE 3.7~. The effect of dietary glucose level, chronic exercise and litter size on milk energy (a) kcaVml1 and b) kcal1day)l.
DlET EXERCISE LlïTER SlZE
120% GLUC rn 40% GLUC 1160% GLUC m
EXERCISED SEDENTARY
m m 8 PUPS Ba12 PUPS
1. Values are least square means (LSM). 1 kcal = 4.18 W. Columns within a group not sharing the same letter are signifkantly dMerent from one another, pc0.05.
FIGURE 3.7d The effect of dietary glucose level, chronic exercise and litter size on 24 hour milk yieldl.
rn 20% GLUC t~ 40% GLUC i 60% GLUC m
EXERCISED SEDENTARY
u 8 PUPS
H 12 PUPS
1. Values are least square means (LSM). Columns within a group not sharing the same letter are significantiy different frorn one another p~0.05.
c TABLE 3.7b Maternel macronutrient intake (gld)' and milk macronutrient output (g/d)2 on Ld15.
D IETARY DIFTARY FAT DIETARY PROTEIN INTAKE INTAKE / MlLK GLUCOSE 1 MlLK PROTEIN PROTEIN (g/d) INTAKE 1 MlLK
DlET (gld) LACTOSE (gld)
20% GLUC 5.1 7 / 2.50 4.83 / 1.46 6.90 / 0.67 (48.47'0) (30.2%) (9.7%)
40% GLUC 5.05 / 2.56 7.41 / 2.40 13.47 1 0.78 (50.7%) (32.4%) (5.8OIo)
60% GLUC 4.89 / 2.46 9.78 1 3.52 19.56 / 0.72 (50.3%) (36.0%) (3.7%)
1. Based on average daily food intake during lactation Fable 3.2). 2. Based on milk macronutrient concentrations*milk yield (kcaVmL) (TableÛJa). 3. Percentages of rnacmnutrient intakes represented as milk macmnutrients are in parentheses.
TABLE 3.8 The effect of materna1 dietary glucose level, chronic exercise and litter size on individual pup welghtl, average daily milk volumelpup*, average daily milk energylpup3 and average daily milk fatlpup* at Ld15.
AVERAGE MILK AVERAGE MlLK AVERAGE MlLK PUP WEIGHT (g) VOLUMEIPUP ENERGY/PUP FATPUP
(rnUd) (kca Vd) (mgld) DlET 20% GLUC 27.63 f 0.52ab 2.24 f 0.1 3a 2.34a 142.13 + 34.65a 40% GLUC 28.29 f 0.51b 2.70 2 0.1 pb 3.l9b 242.93 f 32.35b 60% GLUC 26.21 * 0.53a 2.67 f 0.1 3b 4.44C 373.94 + 36.75c
SEDENTARY 27.25 + 0.42 2.61 1 0.10
IWER SlZE 8 PUPS 29.46 f 0.42b 2.79 f 0.1 1b 3.80b 308.42 i 29.41 12 PUPS 25.30 -1- 0.43a 2.29 f O.lOa 2.72a 197.58 f 27.2ga
TABLE 3.8 CONTINUED.
sIArmmu AVERAGE MlLK AVERAGE MlLK AVERAGE MltK klIwzYw PUP WEIGHT VOLUME/PUP ENERGY/PUP FAT/Pl.JP EFFECT: F / D VALUE F / p VALUE F l p VALUE DlET 4.12 / 0.01 94 3.78 / 0.0282 10.46 / 0.0001 10.46 / 0.0001 EXERCISE O. 17 / 0.6792 1.04 / 0.31 1 1 0.00 / 0.9740 0.04 / 0.8359 D*E 0.68 / 0.5082 0.71 / 0.4963 0.42 1 0.6586 0.89 / 0.41 70 LITTER SlZE 47.83 / 0.0001 10.89 / 0.001 6 9.15 / 0.0038 7.55 / 0.0079
1. Values are weighted least square means (LSM) f SELSM. LSM wRhin a group not sharing the same superscript are significantly different from one another, pe0.05. 2. Values are LSM f SELSM. 3. Values are back transformed LSM (analysis done on log transformed data). 4. D20/40/60 = 20%/40%/60% GLUC diets. E = chronically exercised. S = sedentary.
FIGURE 3.8a The effect of materna1 dietary glucose level, chronic exercise and litter sire on individual Ld15 pup weighti.
1. Values are weighted least square means (LSM). Colurnns within a group not shadng the same letter are significantly different from one another, p~0.05.
FIGURE 3.8b The effect of materna1 dietary glucose level, chronic exercise and litter size on Ld15 a) average daily milk volumdpup~ b) average daily milk energylpup2 and c) average daily milk fatlpup'.
AVERAGE MlLK VOLUME / PUP
AVERAGE MlLK ENERGY I PUP
AVERAGE MlLK FAT I PUP 1. Values are weighted least sqwre meanç (LSM). 1. Values are LSM. 2. Values are back transfomed LSM. 1,2. Columns within a group not sharing the same letter are significandy difierent from one another, p<0.05.
CHAPTER 4 - DISCUSSION
This study was the first to report on the combined effects of dietary
glucose restriction and chronic exercise on lactation performance in rats and
concluded that dietary glucose restriction affected milk composition by
decreasing milk fat concentrations and increasing milk protein concentrations,
and that chronic exercise generally affected the marnmary gland by decreasing
the percentage of mammary gland fat. This study in particular is unique in that it
is the first to show that chronic maternal exercise increased milk fat
concentrations and that an interaction between diet and exercise modified milk
lactose concentrations such that exercise in rats fed a low carbohydrate diet
(20% glucose) significantly decreased milk lactose concentrations compared to
a medium (40%) or a high (60%) carbohydrate diet suggesting that in an
exercising rat 20% glucose may not be optimal for adequate lactation. In
addition, the impact of maternal dietary glucose restriction on pup growth in the
first 15 days of life with the heaviest pups being nursed by dams fed a moderate
level of carbohydrate (40% glucose) suggests that the NRC requirement (1978,
1995) for a high level of dietary carbohydrate (~60%) is not optimal for the
lactating rat.
Chronic exercise throughout pregnancy and lactation resulted in
increased milk fat concentrations in Our study. The effects of exercise during
lactation have been investigated by others in both animal (Karasawa, 1981;
Treadway and Lederman, 1986) and human studies (Lovelady et al., 1990;
Dewey et al., 1994). Two animal studies on exercise during pregnancy and
lactation found no change in milk fat concentrations of Wistar rats (Treadway
and Lederrnan, 1986) or mice (Karasawa, 1981). Although mice, not rats were
used in the study by Karasawa (1981), and a different strain of rat (Wistar vs.
Sprague-Dawley) was used by Treadway and Lederman (1986), given the
similarity of the lactation process across mammalian species, the difference in
the effect of exercise on milk fat between these studies and ours probably does
not stem from species or strain differences. Explanation for the difference
between Our study and the previous two reports is probably more related to the
type, intensity and duration of exercise. The intensity and duration of the
exercise in the study by Karasawa (1981) may not have been different enough
from the control group to show an effect on milk fat since al1 exercise was
voluntary. In addition, the study by Treadway and Lederman (1986), subjected
rats to swimming exercise in contrast to the treadmill exercise in our study which
may explain why Treadway and Ledeman (1986) found no change in milk fat.
It has been shown that swimming is a good aerobic exercise that increases
physical fitness, but in contrast to more weight-bearing forms of aerobic
exercise such as running or cycling it does not effectively promote weight loss
or body fat loss (Gwinup, 1987; Caldwell, 1988). Swirnming exercise in the
study by Treadway and Lederman (1986) also did not decrease materna1 body
weight cornpared to the sedentary group; however, chronic running exercise in
our study lead to decreased weight gain during pregnancy and final body
weight at the end of pregnancy as well as decreased mammary gland fat on day
15 of lactation al1 of which rnay have mediated changes in milk fat. (The
difference in final body weights between the exercised and sedentary dams is
not due to differing litter sizes or litter size weights as the number of pups born
to dams and the litter weights per dam were not significantly different between
the exercise and sedentary groups - Leccisi-Esrey, 1991). Two human studies
(Lovelady et al., 1990; Dewey et al., 1994) have also examined the effect of
exercise on lactation and found no differences in milk fat between exercised
(aerobics) and control groups. The wornen in these studies, however, were
exercised only during the period of lactation and not during both pregnancy and
lactation which could have affected the amount of weight gained and the
amount of fat accumulated in the mammary tissue. Milk fat is derived from two
sources, the circulation and de novo synthesis from glucose within the
mammary gland (Williamson et al., 1984). During lactation there is an increase
in mammary uptake of blood triglycerides and an increase in lipoprotein lipase
(LPL) activity in the mammary gland (Hamosh et al., IWO). In addition, exercise
is known to increase adipose tissue lipolysis (Treadway and Lederman, 1986).
Given the decreased weight, weight gain and mammary gland fat of the
exercised rats in Our study, we suggest that the increase in milk fat may be due
to increased mammary gland and body fat lipolysis leading to increased
substrate for milk fat synthesis. ARhough body fat of the dams was not
measured, it is assumed that the decrease in body weight was largely due to
body fat loss since weight bearing exercise is known to preserve or increase
lean body mass (McArdle, 1986). Exercise would be expected to increase
materna1 plasma levels of non-esterified fatty acids (NEFA) via increased
lipolysis, but since blood samples were not taken irnmediately post-exercise
analysis of plasma NEFA concentration results in Our study not surprisingly
showed no significant difference between exercised and sedentary groups.
A limitation of this study is that the rats were not randomized to the
exercise and sedentary groups but self-selected their activity groups (i.e. those
rats that could run on the treadmill were put into the exercise group). Indeed,
from a purely statistical point of view, not randomizing the rats into activity
groups invalidates the exercise component of this study; however, by
randomizing, there is a risk of some rats in the exercise group not running since
under the best circumstances cornpliance to an exercise regime is difficult to
achieve in pregnant rats. Studies on energy expenditure in exercising pigs
have also not randomized subjects into exercising and non-exercising groups
due to lack of compliance (Personal communication-Dr. H.S. Bayley,
Department of human biology and nutritional science, University of Guelph).
Statistical 'purity' rnay be obtained with randomization, but if the exercise group
is not really exercising then arguably the effect of exercise is not being testedl
Moreover, in the case of non-compliance, any coaxing or prodding of the animal
that does not wish to, or cannot run may introduce a stress factor over and
above that of the actual exercise. In addition, keeping in mind that one of the
ultimate purposes of this area of study is application to human lactation, any
women who exercise during pregnancy and lactation, likely do so voluntariiy.
Our results also showed an important statistical interaction between diet
and exercise on milk lactose concentrations with exercise significantly
decreasing lactose in the milk of rats fed the low carbohydrate (20% glucose)
diet. Previous studies have noted that materna1 dietary energy restriction
(Warman and Rasmussen, 1983; Kliewer and Rasmussen, 1987), dietary
protein restriction (Mansaray and Grimble, 1987), dietary carbohydrate
restriction (Romsos et al., 1 981 ; Koski et al., 1 990; Koski and Hill, 1 990) and
swimming exercise (Treadway and Lederman, 1986), when studied in isolation,
decreased milk lactose concentrations. In the present study, the fact that the
combined effect of exercise and a low carbohydrate diet also decreased milk
lactose concentrations strongly supports the previous suggestion by Treadway
and Lederman (1986) that a decrease in glucose availability to the mammaiy
gland due to the increased energy requirements of exercise was responsible for
the decrease in milk lactose. Our result may illustrate a competition between
the exercising muscle and the mammary gland for glucose since imposing
exercise on rats that may already be at a sub-optimal level with respect to
carbohydrate metabolism may force an increased competition for glucose that
results in lower milk lactose concentrations.
Lactose is considered the osmotic regulator of milk yield (Mepham,
1987); however, in the present study milk lactose was decreased, but milk yield
was not affected by the interaction of diet and exercise. Theoretically, any
decrease in total milk lactose content should cause a decrease in milk yield
such that the concentration of lactose remains unchanged. This does not
appear to be the case across mammalian species since representative
concentrations of milk lactose are different from species to species. For
example, the milk lactose concentration of cows is -50 glL, dogs -45 glL,
humans -60 glL and Sprague-Dawley rats -25-30 glL (Oftedal and Iverson,
1995). It should be noted that cows and humans commonly have single births
and dogs can have multiple births of -6 pups (Romsos, 1981), but rats usually
have larger Mers (average of -7 pups - NRC, 1995). Regression analysis of
total milk lactose contentMay on milk yield/day in the present study showed that
only 59% of the variability in milk yield could be explained by milk lactose
despite the fact that milk is -80% water (Mepham, 1987; Dils, 1989). This
brings into question the extent to which milk lactose determines milk yield in
species that bear large litters such as dogs and rats, and given the range of milk
lactose concentrations across species it is possible that milk lactose is a
stronger determinant of milk yield in species with higher milk lactose
concentrations (cows and humans).
Milk fat concentrations in the present study were also found to be
modified by the level of glucose in the matemal diet. Dietary glucose restriction
resulted in a graded response in milk fat with milk fat decreasing significantly as
dietary glucose decreased. Not surprisingly, dietary glucose within the range of
20% to 60% was found to be predictive of milk fat. A previous study by Lanoue
and Koski (1994) reported that dietary glucose restriction to 12% and 24%
glucose (-35% and -30% fat respectively) significantly decreased milk fat
compared to a high carbohydrate diet of 60% glucose (-16% fat); however, no
difference was found between the milk fat levels in the 12% and 24% glucose
diet groups (Lanoue and Koski, 1994). Our results taken together with those of
Lanoue and Koski (1994) suggest that maternal dietary glucose is critical to milk
fat synthesis even with relatively high dietary fat levels and that a graded
response in milk fat to dietary glucose levels above 20% is possible, but below
a level of 20% dietary glucose, milk fat is not further decreased. A threshold
may exist for dietary carbohydrate below which glucose is diverted to the
mammary gland in order to maintain milk fat synthesis and thus a minimum
level of milk energy for nursing offspring.
Plasma glucose concentration results in Our study confirm the findings of
others, but plasma insulin and lactate dehydrogenase (LDH) concentrations did
not respond to dietary glucose levels as expected. Oddly, the 60% glucose diet
in Our study significantly decreased plasma insulin compared to the 20%
glucose diet. Although our values are similar to those reported by Lanoue and
Koski (1 994), maternal plasma insulin on day 15 post-partum in their study
responded as expected; it was significantly decreased in the 12% glucose
group compared to the 60% glucose group. Inclusion of exercise in our model
may explain why our results do not agree with Lanoue and Koski's (1994);
however, the exact mechanism is not obvious to us. Plasma glucose
concentrations in Our study were not affected by dietary glucose level,
confirming the results of Lanoue and Koski (1994); however, our values were
slightly lower than theirs and this was likely due to the exercise component in
Our study. Plasma LDH activity levels in our study were measured to
approximate plasma lactate and were significantly increased in the 20%
glucose diet group compared to the 40% and 60% glucose diet groups, but
were not affected by exercise. In contrast, although not significantly different,
Romsos et al. (1 981) and Lanoue and Koski (1 994) found a trend towards lower
blood lactate levels of lactating dogs and rats fed low carbohydrate diets in
cornparison to those of animals fed higher carbohydrate diets. In addition, in a
study by Cobrin and Koski (1995), a 0% glucose diet significantly decreased
materna1 blood lactate levels in rat dams compared to 12% or 60% glucose
diets fed during pregnancy. In the same study, materna1 blood lactate was also
decreased by sedentary activity compared to chronic exercise during
pregnancy.
By using isoenergetic composite diets in the present study we cannot be
certain if Our results are due to varying levels of dietary carbohydrate or fat.
Changes in milk composition in this study may be due to both carbohydrate and
fat, although the general nature of physiology leads us to believe that the
changes are primarily carbohydrate driven. As mentioned above, both the
present results and those of Lanoue and Koski (1 994) showed significantly
lower milk fat concentrations from dams fed diets relatively low in dietary
glucose (ranging from 12%-40%) and high in fat (ranging from 22%-35%)
compared to dams fed the control diets of 60% glucose, 14-1 6% fat. Grigor and
Warren (1980) found that matemal diets of 55% carbohydrate, 20% fat (referred
to as 'high fat diets') resulted in significantly lower rates of mammary gland
Iipogenesis cornpared to a fat-free diet (75% carbohydrate, 0% fat), and it was
suggested by Williamson (1980) that this was indicative of decreased glucose
utilization by the rnammaty gland. Given that the diets used by Grigor and
Warren (1980) were also composite diets (a high fat diet is also a low
carbohydrate diet, and a fat-free diet is also a high carbohydrate diet) and food
intake was not controlled for (diets were not isoenergetic) we are left with the
same question-were the resuits carbohydrate driven or fat driven? Since fat is
>2 times more energy dense than carbohydrate (-17 kJ/g vs. -38 kJ/g), more
carbohydrate is required to replace the same quantity (g) of fat. If the changes
in milk fat were driven by dietary fat, the mammary gland would have to be
extremely sensitive to any relatively small changes in dietary fat compared to
dietary carbohydrate. Physiologically, it seems more logical that the mammary
gland would be more sensitive to the larger changes in the quantity of dietary
carbohydrate.
Lactation is the only normal biological process in mammals that will
jeopardize the mother if necessary in order to continue (Bauman and Currie,
1980). Thus if milk energy is compromisad by a decrease in a particular milk
macronutrient, there may be a compensatory response by increasing another
macronutrient. The rat dams in Our study may have tried to compensate for the
energy lost by the decrease in milk lipid that resulted from dietary glucose
restriction by increasing milk protein concentrations. Specifically, as materna1
dietary glucose level decreased from 60% to 40% to 20%, milk fat also
decreased significantly; however the same graded effect of dietary glucose was
not seen in the total milk energy per mL and per day. Milk protein concentration
was significantly increased by a dietary glucose restriction to 20% and
appeared to cornpensate for lost milk fat energy since the resulting milk energy
was not significantly different from that of the 40% glucose diet group. The
suggestion that the mammary gland may try to compensate for loss of milk
energy with milk protein and not milk lactose is supported by the fact that milk
lactose was not changed by dietary glucose restriction. Multiple regression
analysis provides more supporting evidence as dietary glucose was shown to
be predictive of milk protein such that a decrease in dietary glucose from 60% to
40% to 20% resulted in an increase in protein of -7.5 m g h L of milk. The
increase in milk protein was restricted to the 20% glucose diet group even
though both the 20% glucose and the 40% glucose groups had decreased milk
energy. This perhaps indicates that milk resulting from a moderate level of
dietary carbohydrate contains adequate nutrients for offspring growth and
survival. Results from Our study support this as pups nursed by dams fed a
moderate level of dietary carbohydrate (i.e. 40% glucose) weighed significantly
more on day 15 post-parturn than pups nursed by dams fed a high level of
dietary carbohydrate (i.e. 60% glucose).
The NRC requirements (1978, 1995) for metabolizable energy for a
pregnant or lactating rat cal1 for >60% dietary carbohydrate. Historically, a
control diet of -60% glucose and -14% fat was assumed to be adequate for
pregnancy and lactation needs as most pregnancy and lactation studies on rats
that have fed composite diets have used high carbohydrate control diets.
Results from this study bring into question the appropriateness of this
assumption as significantly heavier pup weights on day 15 of lactation were
achieved by a 40% glucose, 22% fat diet (moderate level of carbohydrate and
fat) compared to a 60% glucose, 14% fat diet (high carbohydrate, low fat). In
this context it remains to be seen whether heavier is necessarily healthier and is
a question for future study. The average daily milk volume/pup in the 40% and
60% glucose diet groups were significantly higher compared to the 20%
glucose diet group, and the average daily milk energy/pup and milk fat/pup
increased significantly from the 20% to the 60% glucose diet groups, but the
60% glucose diet fed to dams did not produce the heaviest pups thus
demonstrating that the higher energy intake by the pups was not transferred to
increased body weight. Whether use of the excess energy intake is attributable
to the pups' activity, heat production or some other mechanism is not clear from
this study. Our results may suggest that at least in rat models, a lower
carbohydrate, higher fat matemal diet may be more appropriate for pup growth.
In support of this, Guo and Jen (1995) found that weanling rat pups (22 days
old) of dams that were fed a 40% fat, 30% carbohydrate diet were significantly
heavier than weanlings of dams fed a control diet of 4.5% fat.
Neonatal survival of breeding animals such as dogs may also be
improved with a moderate carbohydrate, moderate fat maternal diet fed
throughout pregnancy and lactation. Evidence of this was reported previously
by Romsos et al. (1981) who fed pregnant beagles what was called a high
carbohydrate diet (44% of energy from carbohydrate) or a low carbohydrate diet
(0% energy from carbohydrate) and found that only 35% of the pups whelped
by bitches fed the low carbohydrate diet were alive at 3 days of age. The
finding in Our study that rat dams fed a rnoderate carbohydrate, moderate fat
diet had heavier pups on day 15 post-partum than dams fed a high
carbohydrate diet is suggestive of the importance of a moderate carbohydrate,
moderate fat maternal diet to offspring survival and supports the findings of
Romsos et al. (1981) as what they categorized as a high carbohydrate (44% of
energy) diet is similar to the moderate carbohydrate (-39% of energy) diet used
in Our study.
It is possible that milk composition and not total milk energy may play a
more important role in offspring growth. A significant positive correlation
between milk fat concentration and weight gain of rat pups was found by Mozes
et al. (1 993) in the first 10 days of life, but not in the second 10 days of life.
Growing pups may not be able to tolerate high concentrations of milk fat as the
intestinal phase of fat digestion in newborn infants is incomplete due to low
pancreatic lipase activity and bile salt levels (Hamosh, 1979; Hamosh et al.,
1984; Hamosh et al., 1994). In further support of this theory, mean pup weight
(-27 g) on day 15 of lactation in Our study was almost identical to that reported
by Treadway and Lederman (1986) despite the dams in their study having
-40% higher amounts of lipid and -60% higher energy in their milk. The
observation in Our study that milk protein increased in the low dietary
carbohydrate group (2OoA glucose), but not the moderate dietary carbohydrate
group (40% glucose), also supports the idea that milk composition and not milk
energy may be more important for offspring growth since it seems that at 40%
dietary glucose a compensatory increase in protein was not apparent as it was
in the 20% glucose group.
Given the similarity of lactation physiology across mammalian species it
is not entirely inconceivable that a higher fat diet may also be appropriate for
human lactation. The diet of Gambian women in West Africa is relatively high in
carbohydrate and low in fat (Hudson, et al., 1980) compared to the typical North
American diet and total energy intake by pregnant and lactating Gambian
women is far below the amounts recommended by the FAONHO (Paul et al.,
1979). In a study by Prentice et al. (1980), supplementing lactating women for
12 months with a -700 kcaVday (relatively high carbohydrate, low fat biscuit) did
not improve milk output, therefore perhaps the amount of fat was still too low in
the diets of these women to improve their lactation performance.
The process of lactation does not offer any special advantage to an
animal yet it places a large demand on its metabolism (Bauman and Currie,
1980). Given the rather large differences in dietary glucose supplied during
lactation in Our study, the total quantities of the resulting milk macronutrients
were remarkably unaltered, thus demonstrating the 'robustness' of lactation and
the ability of the mammary gland to repartition nutrients in order to preserve milk
quality. In the literature, the concept of nutrient partition has generally focused
on dairy came (Bauman and Currie, 1980). Mammary metabolism in ruminants
and rodents represent two extremes on a continuous spectrum (Mepham,
1987), thus the importance of dietary glucose supply to milk lactose and milk fat
synthesis is different in the rat compared to the ruminant since in the ruminant
very little dietary carbohydrate escapes fermentation in the rumen (Larson,
1985). Not al1 nutrients absorbed by the mammary gland appear in the milk, but
are used for cell division, cell growth, storage or catabolic processes (Mepham,
1985). In Our study, nutrient utilkation with respect to milk production appears
to be different among the three diet groups. Table-3.7b shows that on day 15 of
lactation the total amount of milk protein represented a relatively constant
percentage of daily dietary intake at ~ 4 8 ~ 5 0 % for the 3 diet groups; however as
dietary glucose increased from 20% to 60% the total amount of rnilk fat
represented an increasing percentage of dietary fat intake (-30, 32% and 36%)
and the total amount of milk lactose represented a decreasing percentage of
dietary glucose intake (-IO%, 6% and 4%). This may suggest that as dietary
glucose intake increased glucose is shunted more toward fat synthesis than
lactose synthesis; however, labeled glucose studies are required to confirm or
refute this theory.
The results reported in this study generally support previous findings on
the independent effects of materna1 dietary carbohydrate and exercise on the
mammary gland with the exception of the reported values for mammary gland
weight and percentage of fat. Similar to Our findings, another study which
compared 12%, 24% and 60% glucose diets fed throughout pregnancy and
lactation, but which did not include an exercise component found no effect on
rat mammary gland protein, glycogen or fat concentrations (Lanoue and Koski,
1994). Our results also showed that exercise decreased the percentage of
mammary gland fat supporting the finding of Mottola et al. (1986) that exercise
decreased matemal post-partum skin weight (which included mammary glands)
in rats. In contrast to Our study, Lanoue and Koski (1994) reported that a 12%
glucose/35% fat diet fed throughout pregnancy and lactation significantly
increased mammary DNA concentrations, but significantly decreased cell size
(protein:DNA ratio) cornpared to a control diet of 60°h glucose/lô% fat. The less
restrictive diets with respect to carbohydrate used in Our study could explain
why we found no differences in mammary gland DNA concentration or cell size.
Grigor et al. (1984a) reported that rat mammary gland weight was decreased by
a 75% carbohydrate/4% fat diet fed during lactation compared to a lower
carbohydrate (55%), higher fat (20%) diet. The lower fat (4%) diet used by
Grigor et al. (1984) cornpared to our diets may not have supplied enough
substrate for the dam's energy metabolism or milk fat thus necessitating the use
of mammary fat leading to decreased mammary weight. Our values for
mammary gland protein, DNA and glycogen concentrations as well as
protein:DNA ratio were similar to those found by Lanoue and Koski (1994);
however, percentages of mammary gland fat in our study were -2.3 times
greater than those of Lanoue and Koski (1994) even for the control diet of 60%
glucose which was almost identical to the control diet used in Lanoue and
Koskits (1 994) study. The diet compositions, time of diet feeding (gestation
andor lactation) and the rat strain (Sprague-Dawley) in Our study were similar
to that of Lanoue and Koski (1994) therefore, although the mechanism of action
is not clear the inclusion of the exercise component in our study design may
have been responsible for the difference in mammary gland fat percentages.
Mammary gland mass (assumed to be wet weight) reported by Grigor et al.
(1 984) was approximately half that of the mammary glands in Our study. Use of
a different strain of rat (Wistar) by Grigor et al. (1984) andlor the difference in
period (pregnancy andlor lactation) the diets were fed could explain the
discrepancy in mammaty gland weight.
In general, milk composition and energy in Our study were not
dramatically different from the findings of others (Williamson, 1984; Koski et al.,
1990; Koski and Hill, 1990; Fiorotto et al., 1991 ; Lanoue and Koski, 1994). Our
weights with larger litter sizes (Wilde and Kuhn, 1979; Knight and Peaker,
1982). However, we are unaware of any study that showed as we have
increased mammary gland protein concentration with larger litter sizes. In
contrast to Our results, Grigor et al. (1986b) found significant decreases in
mammary DNA concentrations of dams that nursed smaller litter sizes. The
difference in findings between Our studies may be due to the smaller difference
in litter sizes (8 vs. 12 pups) used in Our study compared to that of Grigor et al.
(1 986b) (2 vs. 10 pups).
Pup weight on day 15 of lactation was also higher in Mers of 8 compared
ta Mers of 12 which confirms findings of other studies (Russel, 1980; Knight and
Peaker, 1982; Fiorotto et al., 1991) and is consistent with the idea that pups in
larger Mers have a limited supply of milk available to them. Our results showed
that pups nursed in smaller Mers of 8 consumed significantly more milk volume,
milk energy and milk fat than pups nursed in larger litters of 12. Whether this
difference in pup body weight will persist is questionable given that Wurtman
and Miller (1976) did not find any significant difference in body weight at 21
days post-partum between rat pups nursed in littes of 8 or 12.
Cumulative food intakes and final maternal body weights at the end of
pregnancy and lactation were not influenced by dietary glucose intake;
however, repeated measures ANOVA revealed that dietary glucose level
affected average daily maternal body weight, daily food intake and 2day weight
gain during pregnancy depending on day of pregnancy, and average daily
matemal body weight during lactation. Similar to Our study, Lanoue and Koski
(1994) and Cobrin and Koski (1995) found that rats fed varying levels of
glucose (1 2%, 24% and 60% glucose and 12% and 60% glucose respectively)
during pregnancy did not differ with respect to cumulative food intake; however,
the total quantities of food consumed were lower than those of Our study.
Pregnant rats have been shown to decrease their intakes of dietary fat
compared to virginal controls when fed separate isoenergetic macronutrient
diets (protein, carbohydrate, fat) (Dial and Avery, 1991). Given that the 20%
glucose diet fed in Our study was also the highest in fat, the rats may have
become satiated sooner and thus did not consume as much/day as the rats fed
the 40% or 60% glucose diets. In contrast to Our findings, despite no significant
differences in cumulative food intake Lanoue and Koski (1994) found that rats
fed a 12% glucose diet weighed significantly less at the end of pregnancy than
rats fed 24% or 60% glucose diets, and Cobrin and Koski (1995) reported that
rats fed a 12% glucose diet gained significantly less weight than rats fed a 60%
glucose diet. Final materna1 weight at the end of pregnancy was not affected by
diet in Our study; however, rats fed the 20% glucose diet had lower 2-day weight
gains from day 10 to 16 of pregnancy compared to rats fed the 40% and 60%
glucose diets. At day 18 of pregnancy a graded response in weight gain was
seen with dams fed the 20% GLUC diet gaining the least amount of weight. At
day 20 of pregnancy, weight gains of dams fed either the 40% GLUC diet or the
20% GLUC diet were lower than that of dams fed the 60% GLUC diet (see
Figure 3.4e). The difference in daily food intake between the diet groups may
explain the weight differences in our study since on certain days rats fed the
20% glucose diet consumed significantly less per day than rats fed the 40% or
60% glucose diets during pregnancy. In contrast to pregnancy, on certain days
during lactation, daily food intake of rats fed the 20% and 40% glucose diets
was significantly greater than that of rats fed the 60% glucose diet; however,
matemal weight during lactation did not correspond to daily food intake as there
was no effect of diet on matemal weight throughout lactation. lncreased daily
food consumption of dams fed the 20% and 40% glucose diets was apparently
not used for matemal needs and was used for milk production andor energy for
exercise. As in Our study, Lanoue and Koski (1994) did not find any effect of
dietary glucose level on cumulative food intake or final materna1 body weight at
the end of lactation (day 15).
Cumulative food intake at the end of pregnancy and lactation as well as
final weight at the end of lactation were also not affected by exercise; however,
exercise did affect daily food intake and 2-day weight gain on certain days
during pregnancy as well as final maternal body weight at the end of
pregnancy. Cobrin and Koski (1995) also found no effect of mild or moderate
exercise on cumulative food intake at the end of pregnancy. In the present
study, exercised dams generally consumed less foodlday during the first part of
pregnancy and gained less weighV2 days during the last part of pregnancy
compared to sedentary dams. Final maternal body weight at the end of
pregnancy was lower in the exercised group compared to the sedentary group.
Similarly, Cobrin and Koski (1995) found that moderate exercise (but not mild
exercise) resulted in lower maternal weight gain at the end of pregnancy.
In our study, maternal plasma cortisol and glucose levels were not
affecteci by chronic exercise suggesting that the exercised dams were not more
stressed than the sedentary dams. Moreover, other studies that have
investigated the effect of dietary carbohydrate restriction during lactation have
reported plasma glucose levels in lactating dams that were similar to ours
(Koski and Hill, 1986; Koski and Hill, 1990; Koski et al., 1990). We are not
aware of any studies similar to ours that have reported cortisol levels.
It is difficult to compare exercise protocols across pregnancy and
lactation studies as different methods for characterizing exercise have been
reported. Oxygen consumption or V02rnax and watts of energy expended in
conjunction with frequency, intensity and duration have been used in exercise
and pregnancy studies to characterize exercise intensity as mild, moderate or
strenuous. Oxygen consumption (V02) of non-pregnant rats has been used to
define exercise intensity in several studies (Shepherd and Gollnick, 1976;
Brooks and White, 1978; Bedford et al., 1979); however, oxygen consumption
corresponding to exercise intensity in pregnant animals has not been
established. Mottola (1996) recently reviewed the use of animal models in
exercise and pregnancy studies, but did not address this issue. Wolfe and
Mottola (1993) have previously reported that maternal VOp is significantly
increased with exercise such as treadmill running (i.e. fitness increaseshraining
occurs), but the magnitude of change is thought to be proportional to maternal
weight gain so that VOp expressed as mUkglmin is similar or only slightly
increased during pregnancy compared to the non-pregnant state at the same
speed of running (Wolfe and Mottola, -1993). Animal studies (Mottola et al.,
1986; Mottola et al., 1989) in exercising pregnant animais have used VOzmax
to define exercise as strenuous, moderate or mild; however, given that pregnant
rats gain weight over time, this method may not be very accurate. If VOzmax is
expressed as mUkg/min then weight gain should be accounted for; however,
this would assume that the exercise regime is adjusted as the animal becomes
heavier, i.e. the frequency, intensity andior duration must be adjusted to
maintain the same V02rnax. Pregnancyllactation studies in animals have not
adjusted the exercise protocol throughout pregnancy (Mottola et al., 1986;
Mottola et al., 1989; Cobrin and Koski, 1995). If rats are to be exercised for the
duration of pregnancy and the exercise protocol is characterked as moderate
based on VOzmax values of non-pregnant rats, at the sarne frequency, intensity
and duration, the amount of oxygen consumed at the beginning of pregnancy
will most likely be lower than the amount consumed at the end of pregnancy
when the rats are heavier (assuming there is no training effect). In other words,
the exercise protocol may start out as moderate intensity, but end at strenuous
intensity. Another method of determining exercise intensity is to calculate watts
of energy expended with duration, intensity as well as body weight taken into
account. The confusion lies in the discrepancy among studies with what exactly
constitutes mild, moderate and strenuous exercise. Watts of energy expended
while running can be calculated by an equation that takes into account the rate
of running, the duration of running, the runner's body weight and the degree of
incline at which the running took place (Cobrin and Koski, 1995). Calculation of
the watts of energy expendedlday by exercised pregnant rats in a study by
Mottola et al. (1983) revealed that -233 W of energy/day were expended at the
beginning of pregnancy and this was characterized as mild intensity aerobic
exercise. A study by Cobrin and Koski (1995), reported that 16 W and 28 W of
energylday were expended by exercising pregnant rats following mild and
moderate exercise protocols respectively. The watts of energy expended by the
rats in Mottola et a1.k (1983) study are about 10 times that of Cobrin and Koski's
(1995), yet Mottola et al. (1983) define their exercise as mild and Cobrin and
Koski (1995) define thein as mild-moderate. The watts of energy expended by
pregnant rats in Our study (-77 Wtd) which was characterized as moderate by
the VOzmax method are in line with energy expenditures reported by Cobrin
and Koski (1995), and can actually be considered high intensity
exercise;however, cornparison to the calculated energy expenditure in Mottola
et al.% (1983) study would lead us to believe that our exercise intensity was not
very strenuous.
Regardless of how the exercise intensity in our study is categorked, we
showed that chronic exercise increased milk fat concentrations which may be
mediated by body and mammary gland fat reserves and that competition
between the mammaiy gland and exercising muscle for glucose exists at low
carbohydrate levels. Our results also showed that matemal dietary glucose
intake >20% produced graded levels of milk fat, and that a moderate
carbohydrate (40% glucose), moderate fat diet rnay be more appropriate for
optimal pup growth. The findings of Our study rnay have significance in three
arenas; laboratory animal science, veterinary science and human nutrition.
With respect to animal diets in lactation studies we suggest that the NRC
requirements for carbohydrate in the lactating rat rnay be too high for optimum
lactation performance and that a more moderate level of maternai dietary
carbohydrate and higher level of dietary fat than what is traditionally fed to dams
rnay be more appropriate for pup growth. In addition, moderate carbohydrate,
moderate fat diets rnay improve neonatal survival rates of breeding animals
such as dogs. Finally, given that the processes of lactation are similar across al1
species even though milk composition rnay differ, it is not inconceivable that
human lactation performance rnay also benefit from a moderate carbohydrate,
moderate fat diet.
Results from this study provide a basis for future investigation. Other
studies using the same diet and exercise model can be conducted in order to
answer metabolic and developmental questions. Analysis of blood sarnples
taken at regular intervals post-exercise will allow the direct effect of exercise on
blood variables to be more closely examined. Liver and muscle glycogen data
as well as glucose tracer studies will allow the question of cornpetition for
glucose between muscle and mammary gland to be more thoroughly
investigated (unfortunately, liver and muscle samples from the present study
were lost in a freezer breakdown). Finally, a longer study rnay answer
questions about pup development: is heavier necessarily healthier and will the
weight difference between 15 day old pups from dams fed a 60% GLUC diet
and a 40% GLUC diet persist into adulthood?
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STATISTICAL METHODS
1. Analysis of Variance (ANOVA). a. Non-transforrned and log transformed data.
proc glm; classes diet exercise litsiz; model MGPro MGGlyc MGFatD MGDNA CellSize MLact MFat MYield BWtP BWtL PGluc PLDH PCort PNEFACFeedP Tkcalml Tkcald MVolpup MEpup MFatpup = litsiz diet exercise diet*exercise; lsmeans diet exercise diet'exercise litsidstderr pdiff;
b. Weighted data.
proc glm; classes diet exercise litsiz; model MGWt MGWtB MGFatW CFeedL MPro Plns = litsiz exercise diet diet*exercise; weight MGWtw MGWtBw MGFatWw CFeedlw MProw Plnsw PNEFAw; lsmeans diet exercise diet*exercisa litsizlstderr pdiff;
2. Nested ANOVA. a. Weighted data.
proc mixed; classes diet exercise litsiz dam; model pupwtf = litsiz diet exercise diet*exercise; weight pupwtfw; random dam(diet*exercise litsiz); lsmeans diet exercise diet*exercise litsiz;
3. Analysis of Covariance (ANCOVA). a. Non-transfotmed and transformed data.
proc glm; classes diet exercise litsiz; mode1 MGFatD MGPro MGGlyc MGDNA CellSize MLact MFat TKcalml Tkcald MYield CFeedP BWtP BWtL = litsiz diet exercise diet'exercise PPWt; Ismeans diet exercise diet*exercise litsiz/stderr pdiff;
b. Weighted data.
proc glm; classes diet exercise litsiz; model MGWt (or MGWtB, MGFatW, CFeedL, MPro) = litsiz diet exercise diet*exercise PPWt; weight MGWtw (or MGWtBw, MGFatWw, CFeedL, MProw); lsmeans diet exercise diet*exercise litsizdstderr pdiff;
c. Nested analysis.
proc rnixed; classes diet exercise litsiz; model AvPupwt = litsiz diet exercise diet'exercise MYield (or MPro, MFat, MLact, Tkcalml, Tkcald, CFeedP or CFeedL); weight AvPupW; random dam(diet*exercise litsiz); Ismeans diet exercise diet*exercise litsiz;
4. Repeated measures ANOVA.
proc mixed; class diet exercise litsiz dam day; model wt(*) = litsiz diet exercise diet'exercise day litsiz*day diet'day exercise'day diet*exercise*day/noint ; randome dam; repeatedlsubject = dam; lsmeans diet exercise litsiz diet*exercise litsiz*day diet*day exercise'day diet*exercise*day;
*wt = pregnancy weight pattern (data was log transfomed and 'random dam' statement was not included in the analysis) lactation weight pattern pregnancy weight gain pattern daily pregnancy food intake pattern daily lactation food intake pattern
5. Multiple Regression Analysis
proc reg; model Myield (or MFat, MLact, MPro) = diet exercise litsiz MGWtB MGFatW MGPro MGGlyc CellSize CFeedP CFeedL;
proc reg; model AvPupwt = MPro MF& MLact VolPup;
