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LIBRARY
Michigan State
University

fi—__

 

IIIIIII

  
 
 

This is to certify that the

dissertation entitled

Relationship of Body Fat, Insulin and Selected
Blood Borne Metabolites to Expression of
of Postweaning Estrus in Primiparous SOWS

presented by

Lee Jay Johnston

has been accepted towards fulfillment
of the requirements for

Ph.D. degreein Anlma] SClence

f .

ajor professor

Mew

MSU is an Affirmative Action/Equal Opportunity Institution 0-12771

 

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RELATIONSHIP OF BODY FAT, INSULIN AND SELECTED BLOOD BORNE METABOLITES
TO EXPRESSION OF POSTWEANING ESTRUS IN PRIMIPAROUS SONS

By

Lee Jay Johnston

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Animal Science

1988

 

 

ABSTRACT

RELATIONSHIP OF BODY FAT, INSULIN AND SELECTED BLOOD BORNE METABOLITES
TO EXPRESSION OF POSTNEANING ESTRUS IN PRIMIPAROUS SONS
By
Lee Jay Johnston

Specific physiological factors which control expression of
postweaning estrus have not been identified in sows. Two experiments

were conducted to determine the effects of body fat and handling-

 

induced stress on expression of postweaning estrus. A third
experiment was conducted to determine if exogenous insulin would
shorten the postweaning interval to estrus in restricted—fed sows.

In experiment I, 22 primiparous sows were used to determine if a
minimal threshold of body fat exists below which return to estrus is
delayed. During lactation, sows received 7, 9, 11 or 13 Mcal
metabolizable energy (ME) daily to produce a range of sow body fatness
at weaning. Body fat was estimated at weaning and at first
postweaning estrus by deuterium oxide dilution. Percentage body fat
at weaning (r2=.24) and at first postweaning estrus (r2=.03) accounted
for a small portion of variation in interval from weaning to estrus.
No minimal threshold of body fatness was detected.

In experiment II, 14 primiparous sows received 7 Mcal ME daily
during lactation to induce delayed return to postweaning estrus.

Stress associated with daily restraint and sampling of blood during

 

 

 

 

Lee Jay Johnston

the postweaning period did not influence interval to estrus.

In Experiment III, 26 primiparous sows consumed 7 Mcal ME daily
during lactation which prolonged the weaning to estrus interval.
During the postweaning period, treated sows received an intramuscular
injection of .75 IU insulin per kg body weight once daily. Control
sows were not injected. Exogenous insulin caused a 68% increase in
concentration of insulin in serum and a 15% decrease in concentration
of glucose in plasma of treated sows compared with control sows.
Expression of estrus was delayed unexpectedly in insulin—treated sows
compared with control sows. Concentration of insulin, glucose and
urea nitrogen were not different between sows that diSplayed estrus
and those that were anestrous. In contrast, sows that displayed
estrus had a higher concentration of non-esterified fatty acids (NEFA)
in plasma during the postweaning period than anestrous sows.

I conclude that body fat does not control expression of
postweaning estrus in primiparous sows. Furthermore, elevated levels
of blood borne insulin, glucose and urea nitrogen do not influence
positively return to postweaning estrus. Non—esterified fatty acids
in blood or some factor related to NEFA appear to have a positive

effect on expression of postweaning estrus.

 

 

ACKNOWLEDGEMENTS

There were many people that helped me tremendously in my graduate
studies. I would like to acknowledge the contributions of these
people. My major professor, Dr. Elwyn Miller, was very patient with
me and open-minded throughout my program. He permitted me to be
involved in a variety of activities which at times distracted me from
my research efforts but he recognized the educational value of these
extra-curricular activities. I would also like to thank him for his
undying interest in my academic development.

The other members of my guidance committee, Drs. R.L. Fogwell,
D.R. Romsos, D.E. Ullrey and N.K. Ames, played many different roles.
They provided academic challenges which improved my research and were
able to get things out of me that I did not know were in me. Thank
you for your contributions of time and talent.

Dr. M.G. Hogberg, Chairman of the Animal Science Department,
provided administrative support for the Extension Assistant position I
held for 18 months. This experience was truly one of the highlights
of my education at MSU. Thank you, Dr. Hogberg, for this unique
opportunity.

I certainly appreciate Dr. P.K. Ku’s patience in helping me
improve my techniques in the laboratory.

I would like to thank P.D. Matzat, Swine Farm Manager, and R.

Kromer, Farrowing House Manager, for feeding and care of sows involved

my experiments.

The graduate students of the swine group contributed to my
;earch by providing critical review of research projects, labor and
nradery when nothing seemed to work. These students include Steve
decki, Bill Weldon, Ormond MacDougald, Jose Menten, Jerry Shurson
d Lora Foehr. Thank you.

S.A. Zinn deserves particular mention for his patience and
~rsistence in helping me successfully measure insulin in blood of
st.

The speed and professionalism with which Ms. Judy Lentz typed
iis thesis allowed me to concentrate on research. Thank you, Judy,
)r your dedication to your work.

I would like to thank my parents, Edward and A. Kathleen Johnston
nd my sister, Linda Johnston, for their blind support of me in my
raduate studies. Their words of encouragement buoyed me in times of

ustration. They taught me at a young age that when you say, "I
n’t do it" then you most likely won’t complete the task. This
sson has served me well.

Finally and most importantly, I would like to thank my wife,
udy, for her love and understanding. Her unending patience,
specially in the final stages of my program, restricted the anxieties
f graduate study to those associated with academic pursuits not
omestic pressures. Completion of this degree is more meaningful to

e because we did it.

 

TABLE OF CONTENTS

Page
T OF TABLES . . . . ...................... viii
T OF FIGURES .......................... ix
RODUCTION ........................... I
’IEN OF LITERATURE ....................... 5
elationships Between Body Fat and Expression of Estrus ..... 7
Body condition and expression of estrus in farm species. . . 7
Role of body fat in onset of puberty ............ 9
Women athletes and amenorrhea ................ 13
Anorexia nervosa and menstrual dysfunction ......... 19
Summary ........................... 21
Ffects of Restricted Feeding on Reproductive Performance . . . . 21
Restricted feed intake by breeding females ......... 21
Restricted feed intake by breeding sows ........... 23
Potential mechanisms for effects of restricted feeding on
expression of estrus ................... 25
Metabolic effects of restricted feeding in sows ....... 34
Summary ........................... 36
ffects of Insulin on Reproductive Functions ........... 39
Diabetes as a model for effects of insulin on reproduction . 40
In vitro effects of insulin on HPO axis ........... 42
Effects of insulin on estrous cycles of farm animals . . . . 44
Summary ........................... 46
inclusions ........................... 47
IENT I Relationship Between Body Fat and Postweaning Interval
. to Estrus in Primiparous Sows .............. 49
:Introduction .......................... 50
iMaterials and Methods ..................... 51
‘ Animals and management ................... 51
Progesterone assay ..................... 54
Body fat .......................... 55
Statistical analysis .................... 56
Results ............................ 57

vi

 

Discussion. . . . . ........... . . . . . ......
Conclusions . . . ............. . . ........ 73
ERIMENT II Effects of Stress After Lactation on Postweaning
Interval to Estrus of Primiparous Sows ....... 74
Introduction ....................... . . . 75
Materials and Methods ..................... 76
Animals and management ................... 76
Statistical analysis .................... 78
Results ............................ 78
Discussion ........................... 80
Conclusions .......................... 83
PERIMENT III Effects of Exogenous Insulin After Lactation on
Postweaning Interval to Estrus of Primiparous
Sows ....................... 84
Introduction .......................... 85
Materials and Methods ..................... 87
Animals and management ................... 87
Hormone and metabolite assays ................ 89
Statistical analyses .................... 91
Results ............................ 93
3 Discussion ........................... 106
I Conclusions .......................... 116
HERAL DISCUSSION ........................ 117
MMARY AND CONCLUSIONS ...................... 124

ST 0F REFERENCES ........................ 126

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LIST OF TABLES

Page
COMPOSITION AND CALCULATED ANALYSIS OF DIETS ...... 52
DAILY ALLOWANCE 0F FEED AND SELECTED NUTRIENTS FOR
PRIMIPAROUS SONS DURING LACTATION ............ 53
EFFECT OF METABOLIZABLE ENERGY INTAKE ON SON WEIGHT,
BACKFAT DEPTH AND PIG PERFORMANCE ............ 58
EFFECT OF DIETARY ENERGY INTAKE DURING LACTATION 0N SON
BODY FAT AND NEANING TO ESTRUS INTERVAL ......... 59
ENERGY INTAKE AND PERFORMANCE OF PRIMIPAROUS SONS THAT
DISPLAYED SHORT OR LONG INTERVALS TO ESTRUS ....... 64
LACTATIONAL PERFORMANCE OF SONS ASSIGNED T0 CONTROL 0R
STRESSED TREATMENTS DURING THE POSTNEANING PERIOD. . . . 79
FREQUENCY DISTRIBUTION OF NEANING TO ESTRUS INTERVAL AND
OCCURRENCE OF ANESTRUS FOR PRIMIPAROUS SONS SUBJECTED TO
DAILY STRESS ...................... 81

LACTATIONAL PERFORMANCE OF SONS ASSIGNED TO CONTROL OR
INSULIN TREATMENTS ................... 94

EFFECT OF EXOGENOUS INSULIN 0N FREQUENCY DISTRIBUTION OF
NEANING TO ESTRUS INTERVAL AND OCCURRENCE OF ANESTRUS FOR
PRIMIPAROUS SONS .................... 98

. CONCENTRATION OF INSULIN AND SELECTED METABOLITES IN

BLOOD AT NEANING AND NEAR END OF EXPERIMENTAL PERIOD . .104

. MEAN CONCENTRATION OF INSULIN AND SELECTED METABOLITES

IN BLOOD DURING THE POSTNEANING PERIOD ......... 105

. PERFORMANCE OF SONS DURING LACTATION CLASSIFIED

ACCORDING TO INSULIN TREATMENT AND OVARIAN STATUS. . . .107

viii

 

 

gure 1.

gure 2.

gure 3.

gure 4.

Jure 5.

lure 6.

Iure 7.

Jre 10.

tre 11.

LIST OF FIGURES

Page
Relationship between intake of dietary essentials and
change in body condition as a potential controller of
estrus expression .................. 8
Relationships among intake of dietary essentials,
change in body composition and hormonal and metabolic
mediators as potential controllers of estrus
expression ....................... 22
Relationships among low intake of dietary essentials,
body metabolism, change in body composition and
incidence of anestrus. ................ 38
Relationship of maternal body fat of primiparous sows
at weaning to interval from weaning to estrus ..... 60
Relationship of empty body fat of primiparous sows at
first postweaning estrus to interval from weaning to
estrus ......................... 62

Relationship between relative backfat loss during
lactation (percentage loss of 24 h postpartum backfat)
and interval from weaning to estrus .......... 63

Relationship between relative weight loss during
lactation (percentage loss of 24 h postpartum weight)
and interval from weaning to estrus .......... 65

Relationship between daily metabolizable energy (ME)
intake during lactation and interval from weaning to
estrus ......................... 66

Concentration of insulin in serum (panel A) and glucose
in plasma (panel B) of control and insulin—treated sows
during 24 h after injection .............. 95

Concentration of insulin in serum (panel A) and glucose
in plasma (panel B) collected 24 h postinjection during
postweaning period from control and insulin—treated

sows .......................... 97
Concentration of insulin and glucose in blood of
control-cyclic (panel A) and control-acyclic (panel B)
sows during the postweaning period ........... 100

ix

 

 

Figure 12.

Figure 13.

Figure 14.

Figure 15.

Concentration of insulin and glucose in blood of
insulin-cyclic (panel A) and insulin-acyclic (panel B)
sows during the postweaning period ........... 101

Concentration of non-esterified fatty acids (NEFA) and
urea nitrogen in plasma of control—cyclic (panel A) and
control-acyclic (panel B) sows during the postweaning
period ......................... 102

Concentration of non-esterified fatty acids (NEFA) and
urea nitrogen in plasma of insulin—cyclic (panel A) and
insulin—acyclic (panel B) sows during the postweaning
period ......................... 103

Relationships among low intake of dietary essentials,
change in body composition, selected metabolites and
incidence of anestrus ................. 119

 

 

INTRODUCTION

The profitability of swine enterprises involved in farrow-to—
finish or feeder pig production is due largely to the level of
reproductive performance measured as pigs weaned per sow per year
(PSY) achieved in the sow herd. Production and financial records from
310 swine farms in Iowa revealed that net profit per sow is related
positively to PSY (Stevermer, personal communication). A way to
naximize PSY is to decrease time required to produce a litter of pigs.
The reproductive cycle of sows can be divided into three phases:
aregnancy, lactation and rebreeding (postweaning phase).
Theoretically, any or all of these phases could be shortened to
increase PSY.

In reality, gestation length cannot be shortened without a
Eoncurrent reduction in number of live pigs at parturition. Reducing
ength of lactation from 35 to 14 d has been estimated to increase PSY
ty 5 to 7% (Britt, 1986). However, many producers have neither the
igh level of management skills nor the high—cost nursery facilities
yecessary to accommodate very young pigs at weaning. Consequently,
hortening length of lactation may be applicable only to a small
roportion of swine producers. Shortening interval from weaning to
onception can have a positive impact on PSY. Delayed return to
astweaning estrus is a problem in some herds. Less than 60% of sows
isplay estrus by d 18 after weaning (Fahmy et al., 1979; English et
1., 1982).

 

 

 

2

Prompt expression of estrus postweaning and conception will
Hnimize the number of days a sow is unproductive. Return to
ostweaning estrus is complicated by many factors. Nutrition (Reese
t al., 1982; King and Williams, 1984a), parity (Rasbeck, 1969; Clark
t al., 1978), season (Hurtgen et al., 1980), breed (Dyck, 1971; Fahmy
t al., 1979), length of lactation (Svajgr et al., 1974), suckling
ntensity (Stevenson and Britt, 1981) and social interactions
iemsworth et al., 1982) affect postweaning interval to estrus.
)viously, the problem of delayed postweaning estrus cannot be
:tributed to a single cause.

Three factors have magnified the problem of delayed expression of
Istweaning estrus in sows. First, producers with high fixed costs
:sociated with confinement facilities cannot afford to retain
Iproductive sows in their operation. Second, intensive production
'stems dictate strict scheduling of facilities. Often, sows
splaying delayed postweaning estrus may not fit the production
Ihedule. Third, many producers in an attempt to increase weight of
gs at weaning have selected sows that produce large quantities of
lk. Unfortunately, nutrient and energy demands for body functions
1 milk production exceed nutrient and energy intake by the sow.
isequently, body tissues are mobilized to satisfy the demands of
:tation resulting in thin condition of sows at weaning. Thin
Idition at weaning is associated with a prolonged weaning to estrus
:erval (MacLean, 1968; Reese et al., 1984). It has been suggested
reseachers studying aberrant menstrual cycles of women that a
imal threshold of body fat must be maintained for successful

roduction (Frisch and McArthur, 1974). The first objective of this

1I

 

 

'esearch was to determine if a similar minimal threshold of body fat
xists for sows below which interval from weaning to estrus is
rolonged. A second objective was to characterize the relationship
etween body fat at the end of lactation and interval from weaning to
strus.

Low intake of dietary essentials is related to increased
ncidence of delayed postweaning estrus of sows (Reese et al., 1982;
ing and Dunkin, 1986a). Low intake of dietary essentials (restricted
eeding) causes changes in body metabolism (Kornegay et al., 1964;
ahill et al., 1966; Nelssen et al., 1985). These metabolic
ijustments occur concurrent with mobilization of body tissue. Some
1ctor(s) associated with metabolic adjustments induced by restricted
aeding may influence interval from weaning to estrus. These factors
1y act independent of changes in body fat. Insulin is a hormone of
antral importance to body metabolism (Etherton, 1982) which responds
I the animal’s plane of nutrition (Cahill, 1966). Furthermore,
sulin has gonadotrophic effects in females (Poretsky and Kalin,
87). Therefore, one may hypothesize that insulin could shorten the
stweaning interval to estrus of sows displaying nutritionally—
duced delayed return to estrus. The third objective of this
search was to determine effects of exogenous insulin on postweaning
terval to estrus in primiparous sows.

Presently, clinical observations of the relationship among
:tricted feeding, low body fat and interval from weaning to estrus
edominate in the scientific literature. But, cause and effect
ationships have not been established. The overall goal of this
sertation is to increase understanding of nutritionally-induced

ayed return to postweaning estrus in primiparous sows.

 

4
research was to determine if a similar minimal threshold of body fat

exists for sows below which interval from weaning to estrus is
prolonged. A second objective was to characterize the relationship
between body fat at the end of lactation and interval from weaning to
estrus.

Low intake of dietary essentials is related to increased
incidence of delayed postweaning estrus of sows (Reese et al., 1982;
King and Dunkin, 1986a). Low intake of dietary essentials (restricted
feeding) causes changes in body metabolism (Kornegay et al., 1964;
Cahill et al., 1966; Nelssen et al., 1985). These metabolic
adjustments occur concurrent with mobilization of body tissue. Some
factor(s) associated with metabolic adjustments induced by restricted
feeding may influence interval from weaning to estrus. These factors
may act independent of changes in body fat. Insulin is a hormone of
central importance to body metabolism (Etherton, 1982) which responds
to the animal’s plane of nutrition (Cahill, 1966). Furthermore,
insulin has gonadotrophic effects in females (Poretsky and Kalin,
1987). Therefore, one may hypothesize that insulin could shorten the
postweaning interval to estrus of sows displaying nutritionally—
induced delayed return to estrus. The third objective of this
esearch was to determine effects of exogenous insulin on postweaning
'nterval to estrus in primiparous sows.

Presently, clinical observations of the relationship among
estricted feeding, low body fat and interval from weaning to estrus
redominate in the scientific literature. But, cause and effect
elationships have not been established. The overall goal of this

issertation is to increase understanding of nutritionally—induced

 

 

REVIEW OF LITERATURE

mm

 

A goal of swine producers is to maintain breeding sows that will
efficiently produce large numbers of pigs on a regular schedule. In
achieving such a goal, producers optimize opportunity for profit.
Nutrition plays a major role in determining level of reproductive
performance achieved by sows. Studying the relationship between
nutrition and reproduction is similar to a trip through a labyrinth.
One encounters many paths while traveling through a labyrinth: some
lead to a wall while others move one closer to the exit. There are a
multitude of factors related to nutrition alone which may adversely
affect reproduction such as energy and amino acid content of the diet,
level of nutrient intake, extent of body tissue mobilization,
interaction between dietary energy and amino acids and others.
Similarly, reproductive efficiency measured as number of live births
ger breeding female per unit time can be influenced at several
Iifferent points in the cascade of endocrine events which lead to
estrus and ovulation. The cascade of events which result in estrus
*nd ovulation involves endocrine functions of the hypothalamus,
Interior pituitary and ovaries. The complex coordination of these
Ihree endocrine organs alone provides a myriad of points where the
ystem could malfunction resulting in impaired reproduction. Nhen

tudying the interaction of nutrition and reproduction, one is quickly

 

6
overwhelmed by the complexity of the interaction and the myriad of

factors which ultimately influence the end result, live births.

Realizing that there are many variables that one can measure to
assess reproductive function, this review will focus primarily on
expression of estrus and some of the factors which directly influence
this variable. An underlying assumption is that ovulation occurs
associated with behavioral estrus. Nithout estrus and ovulation, the
reproductive process is halted. This review will concentrate on three
factors which may be involved in expression of estrus in postweaning
sows.

First, effects of body fat will be discussed with regard to
expression of estrus. If the nutritional program is inadequate sows
use body nutrient reserves to cover nutritional deficits. Body
nutrient reserves may be viewed as an insurance policy against
‘nutritional inadequacies. However, the insurance policy has finite
Ilimits of protection. If body reserves are relied upon too heavily,
eventually performance of the sow will be reduced. This depression in
sow performance may be manifested as reduced size and(or) weight of
the litter at weaning, delayed return to estrus after weaning and
anestrus. Unfortunately, appetite has not kept pace with the
productive capabilities of genetically improved sows. Therefore,
aroducers rely heavily upon body nutrient reserves which negatively
affects reproductive function. This scenario suggests that body
Iutrient stores may play an important role in maintaining sow
>roductivity under suboptimal nutritional conditions. .Consequently,
effects of fasting and restricted feeding on estrus will be discussed

n the second section of this review. Finally, changes in body

 

 

7
composition and restricted feeding influence body metabolism.
Therefore, some aspect(s) of body metabolism may influence estrus
expression. In this review, potential effects of insulin on estrus
will be discussed. Obviously, body fat, restricted nutrient intake
and insulin are interrelated such that each can influence the others.

But, for clarity of presentation, each will be discussed separately.

Relationship Between Body Fat and Expression of Estrus

This segment of the literature review will concentrate on the
body reserves of energy, namely body fat. It has been suggested that
females must maintain a minimal amount of body fat to ensure continued
reproductive function. This suggests that body fat is a mediator or
controller of estrus expression (Figure 1). Previous reports dealing
with the relationship between body fat and reproductive function can
be divided into four categories which include: 1. relationship
between body condition of livestock species and expression of estrus,
2. role of body fat in onset of puberty in animals and women, 3.
relationship between women athletes and occurrence of amenorrhea and
4. anorexia nervosa in women and menstrual dysfunction. These four
areas have been cited by others as providing evidence that body fat
may be a controller of estrus expression. Nhether or not these four
areas support the hypothesis that body fat is a controller of estrus

expression will be discussed.

- Body condition and estrus expression in farm species.

The relationship between body condition (body fat) and expression

of estrus has been described for many farm species. MacLean (1968,

 

 

 

 

 

Low intake of dietary essentials
relative to reproductive needs

 

 

 

 

LLoss of body fat] Clinical

observation

 

 

High incidence of anestrus “-

 

 

 

Figure 1. Relationship between intake of dietary essentials and
change in body condition as a potential controller of estrus
expression.

 

 

9
1969) reported that sows in thin body condition after lactation

displayed a prolonged interval to first postweaning estrus when
compared with sows in moderate body condition. This observation
caused the authors to coin the phrase "The Thin Sow Syndrome". Sows
that lost excessive amounts of body weight and backfat during
lactation experienced a delayed return to postweaning estrus (Reese et
al., 1982; King and Williams, 1984; King and Dunkin, 1986). As body
condition improved, a greater percentage of dairy cows displayed
estrus by d 24 postpartum (Fulkerson, 1984). Beef cows that
maintained body weight and condition had a shorter postpartum interval
to estrus than cows that lost body condition (Cantrell et al., 1982;
Rutter and Randel, 1984). Similarly, mares entering the breeding
season in thin body condition displayed delayed onset of estrus
compared with mares in moderate body condition (Henneke et al., 1984).

From these studies, it would seem that body fat is related
positively to expression of estrus. These studies provide clinical
observations of the relationship between body fat and expression of
estrus. They do not explain how body fat may influence estrus
expression.

Role of body fat in onset of pgperty.

The physiological changes that occur at onset of puberty are very
similar to physiological changes when ovarian activity is re—
established after periods of lactational anestrus (Haresign et al.,
1983). Consequently, initiation of puberty has been used as a model
to study extended anestrus in farm livestock. Onset of puberty and
associated first estrus or menarche occurs as the culmination of a

complex series of biological events that take place during growth of

 

 

 

 

10
females. As females grow, absolute body weight and weight of body fat

increase. Also, the relative proportion of body fat increases as
growth proceeds (Hafez, 1969). Kirkwood and Aherne (1985) stated
that, "puberty will occur only if the female has achieved a certain
level of physiological maturation".

Severe underfeeding which results in depressed growth rate delays
onset of puberty in gilts (Kirkwood and Aherne, 1985), heifers (Short
and Bellows, 1971), mice (Hansen et al., 1983), rats (Glass et al.,
1979) and girls (Frisch, 1972). These observations imply that
chronological age is not a primary controller of onset of puberty.

Under ad libitum feeding conditions, rats fed a high fat diet

 

displayed vaginal opening (Frisch et al., 1977; Kirtley and Maher,
1979) and first estrus (Frisch et al., 1975) at an earlier age and

 

lighter weight than rats fed a low fat diet. Absolute weight of body
fat was identical for rats receiving high fat and low fat diets;
however, rats fed high fat diets had a greater percentage of body fat
at first estrus (Frisch et al., 1977). One interpretation is that no
critical threshold of relative fatness exists for expression of
pubertal estrus or that if a minimal threshold exists it can be
influenced by diet. Alternatively, the authors suggested that later
maturing rats fed low fat diets gained the same amount of fat as
earlier maturing rats to initiate first estrus. From this
interpretation, they concluded that a minimal threshold of body
fatness must be achieved before puberty will be initiated. In support
of this hypothesis, percentage of body fat was not different between

rats that displayed first estrus at a younger (35 d) compared with an

 

11
older age (46 d; Wilen and Naftolin, 1977). Likewise, estimated I

fatness was not different at vaginal opening of early and late
maturing rats (Kirtley and Maher, 1979).

Restricted-fed rats experienced vaginal opening at an older age,
lighter weight and lower percentage body fat than ad libitum—fed rats
(Glass et al., 1979). In rats, body fatness was correlated positively
to age at puberty (Wilen and Naftolin, 1977). In mice, neither body
weight (Hansen et al., 1983) nor body fat (Hansen et al., 1983;
Perrigo and Bronson, 1983) explained a significant portion of the
variation in age at puberty.

Onset of puberty in farm animals has been studied extensively. A
detailed discussion of factors affecting onset of puberty in gilts is

beyond the scope of this review. The reader is referred to recent

 

reviews by Kirkwood and Aherne (1985) and Christenson (1986). It is
not clear whether restriction of feed during the growing period
hastens or delays onset of puberty in gilts (Kirkwood and Aherne,
1985). If a critical threshold of body weight or fatness were
necessary to initiate puberty, one would expect restricted-fed gilts
to be older at puberty. Lack of a consistent effect of restricted
feeding on age at puberty would suggest that achievement of a minimal
body weight or fat threshold is not necessary to trigger puberty in
gilts. Den Hartog (1984) reported that estimated body fat at puberty
ranged from 20.9 to 38.4%. Body weight at puberty followed a similar
trend. These results do not support the hypothesis that gilts must
reach a minimal threshold of body weight or fat to initiate puberty.
Similarly, dietary manipulation of weight gain in heifers results in

females that experience puberty at different ages and body weights

 

 

12
(Arije and Wiltbank, 1971; Short and Bellows, 1971; McCartor et al.,

1979). Heifers, like gilts, do not seem to have a critical threshold
of body weight which triggers onset of puberty.

Undernutrition suppresses the normal adolescent growth surge in
women (Frisch and Revelle, 1969) and delays menarche (Frisch, 1972).
Menarche occurs when women reach an average critical body weight which
represents a characteristic body composition (Frisch et al., 1973;
Frisch, 1980). Frisch and McArthur (1974) suggested that girls must
reach a minimal weight for height which represents a critical
threshold of body fatness before menarche will occur. This hypothesis
was based on estimates of body fatness derived from the relationship
of body water to height and body weight (Edelman et al., 1952).
Further support of this hypothesis is the observation that prepubertal
ballet dancers possessed a lower percentage body fat and experienced
delayed menarche compared with non—athletic girls of similar age
(Warren, 1980; Cohen et al., 1982).

A biological explanation for a given body fatness to trigger
puberty is based on the close relationship between metabolic rate and
body size. Basal metabolic rate per unit weight declines with
increasing body weight (Brody, 1945). Since body size and fatness are
related positively while body size and metabolic rate are related
inversely, Kennedy and Mitra (1963a) proposed that metabolic rate may
be the signal which triggers onset of puberty. The apparent critical
threshold of body fatness may be an indication of the metabolic rate
necessary to induce puberty. Consequently, body fat would not be a

controller of the onset of puberty per se, but simply a tangential

 

 

13
change which indicates a specific metabolic rate.

Billewicz et al. (1976) argued against the hypothesis of a
critical body weight independent of age as the trigger for menarche.
They observed that mean body weight at menarche was related positively
to age at menarche. This observation would lead one to believe that
body weight is not a controller of menarche.

The effects of body fat on initiation of puberty are equivocal.
Body fat does not seem to be the primary controller of the onset of
puberty. Instead, body fat may be related to some other mediator
which does control induction of puberty.

Women athletes and amenorrhea.

Studies of menstrual cycles in women athletes must be interpreted
in light of the method of data collection. Questionnaires that rely
on recall of the subject are used to collect data on menstrual cycles.
Training intensity is reported by the subject and was not verified
by researchers. Consequently, there is potential for inaccuracy in
data collection which may influence final conclusions. In many
studies researchers relate changes in body weight to characteristics
of the menstrual cycle. These researchers make the assumption that
changes in body fat parallel changes in body weight. This assumption
may not be valid. Realizing these pitfalls do exist, one must use
studies of women athletes and amenorrhea to serve as a guide for
designing controlled studies in livestock species, not to draw
definitive conclusions concerning relationship of body fat to
reproductive cycles. Athletic women have a greater incidence of
amenorrhea (lack of menstrual cycles) and oligomenorrhea (irregular

menstrual cycles and(or) infrequent menstruation) than non-athletic

 

 

14
women (Feicht et al., 1978; Dale et al., 1979; Ronkainen et al.,

1984). Body weight of women who ran more than 30 miles per week was
less and these women had fewer menses per year than non—athletic
women (Dale et al., 1979). Ballet dancers that experienced amenorrhea
had lower body weight than eumenorrheic (regular menstrual cycles)
dancers (Frisch et al., 1980; Cohen et al., 1982).

Weight loss of the magnitude reported to cause amenorrhea would
be composed primarily of adipose tissue loss (Frisch, 1988). Several
authors have proposed that body fat plays a primary role in
determining regularity of menstrual cycles (Frisch and McArthur, 1974;
Dale et al., 1979; Frisch, 1980; Wentz, 1980). Frisch and McArthur
(1974) reported that a minimal threshold of body fat (17%) must be
maintained to ensure regularity of menstrual cycles. An underlying
assumption is that athletic women are leaner than non—athletic women
(Dale et al., 1979) and therefore suffer from a higher incidence of
menstrual disorders compared with the general female population. If
one considers only athletic women, body fat and occurrence of
amenorrhea seem to be related negatively (Dale et al., 1979; Frisch et
al., 1980; Sanborn et al., 1982; Glass et al., 1987). In contrast, no
relationship between body fat and regularity of menstrual cycles was
reported for athletic women by other researchers (Baker et al., 1981;
Wakat et al., 1982). I

The physiological importance of body fat in maintaining regular
menstrual cycles has not been elucidated. From a long term
perspective, Frisch (1988) suggested that control of menstrual cycles

by body fat is an evolutionary adaptation. This adaptation ensures

 

 

 

15
that adequate energy reserves are available to maintain pregnancy and

early lactation. Frisch (1988) suggests that this adaptation would
guarantee survival of the species in times of low food supply. From a
short term view, body fat can be a significant source of extragonadal
estrogens. Body fat has been shown to aromatize circulating androgens
to estrogens (Nimrod and Ryan, 1975). Estrogen participates in
feedback mechanisms which influence release of gonadotropic hormones.
Loss of body fat represents a loss of estrogen producing tissue.
Amenorrheic athletes had less body fat and lower serum estradiol
concentrations than eumenorrheic athletes in the follicular phase of
their cycle (Glass et al., 1987). Female runners were leaner and had
higher serum testosterone concentrations than non—active women (Dale
et al., 1979). But, serum estradiol was not different between the two
groups. This may suggest decreased extragonadal aromatization of
androgens to estrogens. Kirkwood and Aherne (1985) suggested that
anestrus in females with low body fat may be due to reduced
extragonadal production of estrogens in adipose tissue resulting in
reduced plasma estrogen concentration. The physiological relevance of
this hypothesis has not been established. It is not clear if body fat
contributes a significant portion of the total estrogens found in
plasma. Secondly, ovarian compensation may occur when body fat is low
to prevent reduction of plasma estrogen concentrations.

Determination of body fat in human subjects is indirect and
inaccurate. Many researchers have used the relationship between body
weight and total body water (Frisch and McArthur, 1974; Wentz, 1980;
Sanborn et al., 1982) to predict body fatness. Total body water and

body fat are related inversely (Edelman et al., 1952). Equations were

 

16
developed from isotope dilution studies that estimate body water as a

function of weight (Edelman et al., 1952) or weight and height
(Mellits and Cheek, 1970). Other workers used skinfold thickness as a
measure of body fat (Dale et al., 1979; Glass et al., 1987). Skinfold
thickness is a non-invasive, easily obtainable measurement.
Unfortunately, the skinfold technique may not be accurate enough for
measurement of body fat in any one individual (Lutter and Cushman,
1982; Butte et al., 1985). Consequently, studies which predict body
fatness from height and weight or skinfold thickness should be
interpreted cautiously. A more accurate but difficult measurement of
body fat can be obtained by underwater (hydrostatic) weighing (Behnke
and Wilmore, 1974). Using hydrostatic weighing, Calberg et al. (1983)
found that oligomenorrheic and amenorrheic athletes had 3% less body
fat than eumenorrheic athletes. In contrast, no difference in body

fat determined by underwater weighing was observed between amenorrheic
and eumenorrheic distance runners (Sanborn et al., 1987).

In addition to being lower in body weight and leaner, athletic
women are more active than non-athletic women. Increased incidence of
amenorrhea and oligomenorrhea observed in female athletes may be a
direct effect of exercise. In such a scenario, changes in body weight
and fat may not cause menstrual irregularity but may be results of
exercise. In support of this hypothesis, incidence of amenorrhea is
associated positively with number of miles run in training each week
(Feicht et al., 1978; Lutter and Cushman, 1982; Sanborn et al., 1982).
In other studies, number of training miles run each week did not

influence incidence of menstrual dysfunction (Glass et al., 1979;

 

 

l7
Shangold and Levine, 1982; Glass et al., 1987). Different responses

to training mileage may be explained by different nutrient intakes of
experimental subjects. Unfortunately, these researchers did not
report daily intake of energy or nutrients.

Professional ballet dancers have a higher incidence of menstrual
dysfunction than sedentary women (Frisch et al., 1980; Warren, 1980;
Cohen et al., 1982). Among ballet dancers, amenorrheic women are
leaner than eumenorrheic women (Frisch et al., 1980). Low body weight
was associated with amenorrhea (Frisch et al., 1980; Cohen et al.,
1982) and delayed menarche (Warren, 1980) in ballet dancers. In
amenorrheic women, regular menstrual cycles returned with gain of body
weight (Frisch, 1980). This observation would suggest that body
weight may control menstrual cycles in women. However, interruption
of training in amenorrheic dancers resulted in regular menstrual
cycles without gain in body weight or change in body fatness (Warren,
1980). Return to exercise resulted in reversion to amenorrhea without
change in weight. Warren (1980) suggested that energy balance may
play an important role in controlling menstrual cycles of ballet
dancers. Exercise disrupted regular menstrual cycles in non—athletic
women (Bullen et al., 1985). Furthermore, exercise-induced weight
loss caused a greater incidence of delayed menses which worsened as
exercise continued compared with women who maintained constant body
weight during exercise. Female rats subjected to daily exercise
experienced lower body weight gains and higher incidence of irregular
estrous cycles than did non—exercised controls (Carlberg and Fregly,
1985). Forced swimming reduced body weight and body fat and disrupted

estrous cycles of rats (Axelson, 1987).

 

 

 

18

Exercise alters reproductive cycles of humans and rats. Are the
negative effects of exercise due to lower energy balance? Obviously,
exercising women and rats that experience depressed weight gain are in
reduced energy balance. Possibly, some factor(s) associated with
energy balance controls the reproductive cycle. Weight loss, fat loss
and reduced weight gain are caused by changes in energy balance and
may not be related causally to irregularities of the reproductive
cycle.

Several researchers have investigated endocrine aspects of
amenorrheic athletes and reported depressed circulating levels of
follicle stimulating hormone (FSH; Dale et al., 1979; Warren, 1980)
and(or) luteinizing hormone (LH; Dale et al., 1979; Warren, 1980;
Baker et al., 1981; Bullen et al., 1985; Reizer, 1986) associated with
athletic amenorrhea. In association with low circulating
gonadotropins, serum estradiol was depressed in athletic women
compared with controls (Dale et al., 1979; Ronkainen, 1985). Moderate
exercise has been shown to suppress peak progesterone concentration in
saliva during the luteal phase of the cycle (Ellison et al., 1985).
Bullen et al. (1985) reported abnormal luteal function and loss of LH
surge in exercising women. If exercising women lost weight, incidence
of abnormal luteal function and loss of LH surge was associated with
increased frequency of exercise. In amenorrheic ballet dancers,
patterns of gonadotropin release resembled gonadotropic release of
premenarchial women (Warren, 1980). Reduction of LH was more severe
than reduction of FSH. Endurance runners displayed blunted LH

response to exogenous gonadotropin releasing hormone (GnRH) compared

 

 

19
with control women (Ronkainen, 1985). Similarly, interval to GnRH-

induced peak concentration of LH was extended significantly by simple
weight loss (Vigersky et al., 1977).

Athletic amenorrhea seems to originate from the hypothalamic-
pituitary axis rather than the ovary (Keizer, 1986). Exercise-induced
depression of LH concentration wanes several hours after exercise and
returns to normal (Keizer, 1986). If frequency or duration of
exercise is high, normal follicular growth may be reduced resulting in
aberrant menstrual cycles. Aberrant ovarian function cannot be ruled
out but it would appear that athletic amenorrhea results at least in
part from abnormal function of the hypothalamic-pituitary axis.
Anorexia nervosa and menstrual dysfunction.

Anorexia nervosa (AN) is a cachexic condition characterized by
severe weight loss, hypothermia, constipation and amenorrhea (Warren
and Vande Wiele, 1973). The cause of AN is thought to be
psychological in nature. Nonetheless, AN has been used as a model to
investigate the relationship between loss of body weight and
amenorrhea. Severe weight loss caused by AN depressed concentrations
of FSH and LH in plasma (Lundberg et al., 1972; Vigersky et al.,
1977). Other researchers reported a depression of LH but normal FSH
concentrations in AN patients (Danowski et al., 1972; Warren and Vande
Wiele, 1973; Warren et al., 1975). Depressed concentration of
gonadotropins in plasma seems responsible for amenorrhea in patients
with AN. It is not clear what factor(s) is responsible for depressed
gonadotropin concentrations. Depressed secretion of gonadotropins in
response to exogenous GnRH has been demonstrated in AN patients

(Warren et al., 1975). These findings indicate impairment in

 

 

 

 

20
synthesis and(or) release of gonadotropins by the anterior pituitary

or reduced binding of LHRH by the anterior pituitary. In contrast,
other workers found no aberration of pituitary function in women with
AN (Lundberg et al., 1972; Wiegelmann and Sollack, 1972). Selective
depression of concentration in plasma LH with no apparent affect on
FSH suggests a different control mechanism for secretion of LH and
FSH. Differential control of LH and FSH secretion has been
demonstrated previously in humans (Rolland et al., 1975) and sows
(Stevenson et al., 1981).

Concentrations of gonadotropins in plasma rise and return to
normal with weight gain in AN patients; however, amenorrhea persisted
in 47% of cases (Warren and Vande Wiele, 1973). Similarly,
gonadotropin secretion in response to exogenous GnRH improved with
weight gain (Warren et al., 1975). These observations suggest that
weight gain or some weight related factor is very important to elicit
normal profiles of gonadotropins in women with AN.

Although similarities exist between amenorrhea caused by AN and
that caused by loss of weight and body fat, the AN model for
amenorrhea may be confounded by psychological factors. In many
instances, amenorrhea observed with AN precedes weight loss (Danowski
et al., 1972; Lundberg et al., 1972; Warren and Vande Wiele, 1973).
This would suggest that amenorrhea associated with AN is caused by
factors other than body weight loss. In further support of this

hypothesis, amenorrhea persisted after weight gain and the return to
normal gonadotropin profiles in 47% of women recovering from AN

(Warren and Vande Wiele, 1973). Possibly, psychological aspects of AN

 

 

 

 

21
play a very important role in controlling menstrual cycles and

therefore limit the usefulness of AN as a model to study relationship
of body fat and reproductive cycles.
Mum:-

Based on the four lines of evidence reviewed in this paper, one
cannot state definitively that low body fat is a causative factor in
amenorrhea or anestrus of reproducing females.‘ However, low body fat
is associated with aberrant menstrual and estrous cycles. Since loss
of body weight and body fat are reflective of changes in energy
balance, one may hypothesize that hormonal and(or) metabolic factors
associated with change in energy balance may be more central to
control of menstrual and estrous cycles. Consequently, the simplified
model of anestrus in livestock becomes more complicated by including
other hormonal and metabolic factors related to loss of body fat which

may influence return to estrus (Figure 2).

Effects of Restricted Feeding on Reproductive Performance

In the previous section of this review, it was suggested that
changes in body fat are reflective of energy balance. One may easily
hypothesize that nutrient balance directly influences hormonal or
metabolic factors which ultimately influence return to estrus. In
this scenario, changes in body fat would be tangential and may or may
not be involved in controlling return to estrus (Figure 2). This
section will focus on effects of restricted feeding on reproductive
performance and body metabolism.

Restricted feed intake by breeding females

Dietary restriction depresses reproductive performance. Reduced

 

 

 

22

 

 

Low intake of dietary essentials
relative to reproductive needs

 

 

/ \’

 

 

Loss of body
fat

 

 

 

?
===>

——I

 

 

Hormonal and
metabolic mediators

 

 

\/

 

 

High incidence of anestrus

 

 

 

Clinical
observation

Figure 2. Relationships among intake of dietary essentials, change
in body composition and hormonal and metabolic mediators as
potential controllers of estrus expression.

 

23
reproductive performance is manifested primarily as a failure to

express estrus and subsequently, a reduced pregnancy rate. A 50%
reduction in intake of total digestible nutrients (TDN) during late
gestation and lactation resulted in a 75% reduction in pregnancy rate
of mature beef cows (Wiltbank et al., 1962; Wiltbank et al., 1964).
This reduction in pregnancy rate was due to failure of energy-
restricted cows to display estrus. Gauthier et al. (1983) reported
that a 25% reduction in dietary energy and nitrogen intake decreased
number of cows displaying estrus during the first 45 days of
lactation. Severe to moderate restriction of dietary energy and
protein blocked estrous cycles of beef heifers (Bond et al., 1958;
Hill et al., 1970). Similarly, restriction of dietary energy intake
during late gestation and lactation (Dunn et al., 1969) or during
estrous cycles (Spitzer et al., 1978) lowered the proportion of
females expressing estrus. Average energy balance during the first 20
days of lactation was related inversely to interval from parturition
to first ovulation in Holstein cows (Butler et al., 1981).

Restricted feed intake bv breeding sows.

Feed intake of sows during lactation has a profound effect on
postweaning expression of estrus. Lactational feed intake is related
negatively to postweaning interval to estrus (Hughes and Calder, 1979;
King and Williams, 1984a; King and Dunkin, 1986a). Implicit in these
findings is that increased intake of energy and protein shortens
postweaning interval to estrus. One may ask which dietary essentials
are responsible for hastening return to estrus? Sows fed 8 Mcal

metabolizable energy (ME) daily during lactation experienced delayed

 

 

24

return to estrus compared with sows fed 16 Mcal ME (Reese et al.,
1982). No difference in percentage of sows that displayed estrus by
day 14 postweaning was observed when lactational energy intake ranged
from 10 to 14 Mcal ME per day (Nelssen et al., 1985).

Several workers have reported that increasing dietary protein
intake during lactation shortened intervals to postweaning estrus
(O’Grady and Hanrahan, 1975; King and Williams, 1984b; Brendemuhl et
al., 1987). King and Dunkin (1986b) found lactational ME intakes of
10 to 14.5 Mcal daily had no effect on interval to postweaning estrus
but increasing protein intake from 508 g to 815 g daily reduced
interval to estrus. These researchers suggested that there appears to
be a critical energy level of 10 Mcal ME per day during lactation
below which weaning to estrus interval is extended independent of
protein intake. Above the critical energy level, protein intake seems
to play a more important role in controlling postweaning interval to
estrus. Restricting energy intake to 8 Meal ME daily during
lactation had no significant effect on weaning to estrus interval;
however, lactational protein intake was associated negatively with
postweaning interval to estrus (Brendemuhl et al., 1987).

Effects of feeding levels during the postweaning period on
interval to estrus have been studied extensively. Increasing or
decreasing feed intake during the postweaning period had no consistent
effect on interval to first postweaning estrus (Aherne and Kirkwood,
1985). Evidently, nutritional and metabolic events that occur during

lactation control the interval from weaning to estrus.

 

 

 

25
Potential mechanisms for effects of restricted feeding on expression
of estrus.

Moderate to severe restriction of dietary energy and(or) protein
during lactation prolonged interval from weaning to estrus. But, the
mechanism of this effect is not clear. Two possible hypotheses are
presented below. First, body protein or fat may influence interval to
estrus directly. Alternatively, restricted intake of dietary
essentials may directly influence hormonal or metabolic factors which
control return to estrus.

In addressing the former hypothesis, numerous studies have
demonstrated a negative relationship between energy and(or) protein
intake during lactation and loss of body weight in sows (Hitchcock et
al., 1971; Reese et al., 1982a, Nelssen et al., 1985; Johnston et al.,
1986), rats (Glore and Layman, 1985), heifers (Spitzer et al., 1978)
and ewes (Haresign, 1981). In many studies, there was a positive
relationship between loss of body weight and interval to estrus (King
and Williams, 1984a,b; Kirkwood et al., 1987a,b). Weight loss may not
be as important as composition of weight loss when considering effects
on interval to estrus. Sows restricted to 8 Mcal ME daily during
lactation with adequate protein had less backfat and flank fat than
sows fed 16 Mcal while loin eye area and various muscle weights were
not affected by energy restriction (Roos et al., 1987). A 50%
reduction in both energy and protein intake during lactation resulted
in loss of adipose tissue and muscle mass of multiparous sows (Etienne
et al., 1982). Both adipose and muscle tissue can be catabolized

during lactation in the face of dietary restriction. Sows fed high

 

 

 

26
energy, low protein diets during lactation lost similar total body

weight but less backfat than sows fed low energy, high protein diets
(King and Williams, 1984b; Brendemuhl et al., 1987). Thus,
composition of diet can influence relative proportion of fat and
protein lost in a given amount of body weight.

Bpgy protein. Researchers have questioned what aspect of body
weight loss may be related to postweaning interval to estrus.
Investigations have concentrated on loss of lipid (adipose tissue) and
protein (muscle tissue) as possible controllers of interval from
weaning to estrus. Effects of body fat on fertility and expression of
estrus have been addressed in a previous section of this review.

Absolute level of body protein after lactation or relative loss
of body protein during lactation may control interval from weaning to
estrus (King, 1987). Several lines of evidence support this
hypothesis. Improved nitrogen balance due to increased protein intake
during lactation shortened weaning to estrus intervals (King and
Williams, 1984b; King and Dunkin, 1986b). King and Dunkin (1986b)
reported that age and weight of primiparous sows at farrowing were
related negatively to interval from weaning to estrus. Similarly,
multiparous sows are heavier than primiparous sows and therefore have
a greater mass of body tissue which can be mobilized to ensure prompt
return to estrus. Multiparous sows have a shorter weaning to estrus
interval than primiparous sows (Rasbeck, 1969; Hurtgen et al., 1980).
Although older, heavier sows generally have a greater mass of body
protein then younger, lighter sows, one can not conclude that the
negative relationship between interval to estrus and age or body

weight is due to body protein. Other factors associated with

 

 

 

27

increasing age or body weight that may influence interval to estrus.

Furthermore, maternal body size and muscle mass of primiparous
sows increase during pregnancy and lactation and therefore these sows
may have a higher relative requirement for essential amino acids
compared with multiparous females. Low lysine diets seem to have a
greater negative effect on interval to estrus in primiparous sows
compared with multiparous sows (O’Grady and Hanrahan, 1975). In a
summary of eleven experiments, King (1987) reported that protein
intake during lactation accounted for 68% of variation in interval
from weaning to estrus. Estimated body protein at weaning or
estimated loss of body protein during lactation explained 63% of
variation in interval to estrus. Body protein had a more pronounced
effect on interval to estrus than body weight or body fat. One must
regard these findings cautiously because estimates of body composition
were based on live weight and ultrasonically determined backfat depth
(King et al., 1986). A direct test of effects of body protein on
interval to estrus is necessary before definitive answers can be
achieved.

Hormonal and metabolic factors. Another hypothesis is that
restricted intake of dietary essentials may directly influence
hormonal or metabolic factors which control return to estrus. These
direct effects of diet on hormonal or metabolic factors may be
independent of changes in body composition. Before one can
investigate any aberrations in the metabolic and(or) hormonal milieu
which may be present in females with delayed return to estrus, one

must understand the normal sequence of events that leads to

 

 

 

28
postweaning estrus. The reader is referred to recent reviews of
postpartum endocrinology of the sow (Edwards, 1982; Britt et al.,
1985). It is not the intent of this review to duplicate work done so
skillfully by previous authors. However, a few aspects of the
hormonal milieu in postpartum sows need to be highlighted to enhance
the reader’s understanding of later discussions.

The cascade of events which directly influence estrous cycles
involves coordination among the hypothalamus, anterior pituitary and
ovaries. Each of these structures synthesize and release hormones
that coordinate events which result in follicular growth, estrus and
ovulation. Briefly, the cascade of events is circular in nature but is
conceptually thought of as beginning in the hypothalamus. The
hypothalamus synthesizes and releases luteinizing hormone releasing
hormone (LHRH) into the hypophyseal portal circulation in a pulsatile
fashion. This portal blood system carries LHRH to the anterior
pituitary where LHRH stimulates synthesis and pulsatile release of
FSH and LH.

The gonadotropins (FSH and LH) are released into the systemic
circulation to be transported to the ovaries. At the ovaries, FSH and
LH stimulate growth of ovarian follicles. Growing ovarian follicles
produce steroid hormones, primarily estradiol, which are released into
peripheral blood thus reflecting follicular growth. Rising estradiol
concentration in peripheral blood feeds back on the hypothalamus and
anterior pituitary to control release of LHRH and gonadotropins.
Eventually, estradiol levels rise to a peak concentration which is
associated with signs of behavioral estrus and the preovulatory surge

in LH release (Hansel and Convey, 1983). Ovulation occurs

 

 

 

 

29
approximately 24 to 48 h after peak LH concentrations. This brief
overview outlines events that occur in sows with a short interval to
estrus. These events begin with elevated gonadotropin concentrations
in peripheral blood in late lactation and culminate with estrus and
ovulation about five to seven days postweaning.

When investigating the problem of delayed return to postweaning
estrus, one can identify several points where hormonal and(or)
metabolic factors may impair the hypothalamic—pituitary—ovarian (HPO)
axis. Impaired release of hormones or decreased responsiveness of
organs at any level in the HPO axis may delay return to estrus or
cause complete anestrus. One may theorize that ovaries of anestrous
sows are not responsive to stimulation by gonadotropins.
Administration of pregnant mares’ serum gonadotropin (PMSG) to
anestrous gilts and primiparous sows resulted in estrus and ovulation
(den Hartog and van der Steen, 1981; King et al., 1982; Britt et al.,
1986). Administration of PMSG on the day of weaning hastened the
return to estrus especially during summer when interval to estrus is
prolonged (Hurtgen and Leman, 1979). Evidently, ovaries of anestrous
sows are responsive to gonadotropin and capable of follicular growth
and steroid production. Impaired release of gonadotropic hormones
from the anterior pituitary may cause the anestrous condition.
Failure of the pituitary to function properly may result from
inadequate synthesis of gonadotropic hormones, depressed pituitary
responsiveness to LHRH, inadequate stimulation of the pituitary from
LHRH, or inability of the pituitary to release gonadotropins. Any

combination of these potential aberrations in pituitary function could

 

 

 

30
be responsible for delayed return to estrus.

Pituitary function has been tested in yiyp via GnRH challenge.
Primiparous sows anestrous for more than 40 days postweaning displayed
estrus after 55 h of estradiol benzoate treatment with a single bolus
injection of GnRH (Cox et al., 1983). Similarly, anestrous

primiparous sows expressed estrus within four days of initiation of

 

hourly pulses of exogenous GnRH (Armstrong and Britt, 1985).

Injection of estradiol benzoate in long—term anestrous sows increased
LH concentration in serum (Cox et al., 1983). From these experiments,
it appears that the anterior pituitary is responsive to GnRH
stimulation and positive feedback from ovarian steriods. However, one
must recognize that in yiyp challenge studies may not yield results
representative of normal physiological conditions. Quantification of
normal concentrations of LHRH in the hypophyseal portal blood is
difficult. Consequently, one does not know if doses of GnRH
administered in previous studies were physiological or pharmacological
doses. Pharmacological doses of GnRH may result in a response of the
pituitary which is not indicative of the pituitary’s physiological
responsiveness to LHRH stimulation.

If ovaries are responsive to stimulation by gonadotropic hormones
and pituitary is responsive to GnRH stimulation then one concludes
that the aberration in function of the HPO axis in sows that display
prolonged interval to estrus may be at the hypothalamus. One may
hypothesize that some metabolite, catabolic end product, metabolic
hormone or combination of these factors affects the hypothalamus to
inhibit the release of LHRH. Suppression of LHRH would effectively

arrest the cascade of events in the HPO axis. This unidentified

 

31
factor(s) or messenger which inhibits LHRH release must also be
associated with the conditions of extensive body tissue catabolism and
depressed intake of dietary essentials consistently observed in sows
with prolonged interval to postweaning estrus.

Researchers have observed a consistent relationship between
undernutrition and reproductive failure of breeding females.
Reproductive failure is manifested primarily as cessation of estrous
cycles. Anestrus resulting from restricted feeding seems to be caused
by reduced concentrations of gonadotropins in serum. Restricted
intake of calories and protein lowered concentration of LH in serum of
rats (Howland, 1971, 1972; Ibrahim and Howland, 1972; Howland and
Ibrahim, 1973), sheep (Haresign, 1981; Foster and Olster, 1985),
cattle (Apgar et al., 1975; Gauthier et al., 1983; Imakawa et al.,
1986a,b), humans (Fichter and Pirke, 1984) and swine (Kirkwood et al.,
1977). In contrast, other researchers have reported that restricting
intake of calories and protein to between 85 and 90% of recommended
levels had no effect on LH secretion in beef cattle (Hill et al.,

1970; Rutter and Randel, 1984). However, cows that lost body
condition postpartum had lower basal LH concentration than cows that
maintained body condition (Rutter and Randel, 1984). Evidently,
dietary restriction to 90% of recommended levels was not severe enough
to be below each cow’s individual requirements for energy and protein.
Restricting dietary energy to 33% of recommended levels with adequate
protein did not influence serum LH concentrations in beef heifers
(Spitzer et al., 1978). This study may suggest that the suppression

of LH secretion observed in dietary restriction may be specific to

 

 

32
dietary protein. Gombe and Hansel (1973) fed Holstein heifers 62% of

recommended TDN allowances and reported that basal LH concentration in
plasma progressively increased while progesterone concentration in
plasma decreased over three estrous cycles compared with heifers fed
100% of recommended TDN allowance. The authors suggested that ovaries
of restricted—fed heifers became refractory to stimulation by LH. It
is not clear why LH concentration in plasma increased in response to
dietary restriction. Contrary to the reduction of LH, FSH secretion
seems to be unaffected by dietary restriction (Howland, 1971; Campbell
et al., 1977, Piacsek, 1987). Therefore, the anestrous condition
caused by dietary restriction seems specific to reduced secretion of
LH. This differential effect of restricted feeding on LH and FSH is
consistent with the observation that secretion of LH and FSH is
subject to different control mechanisms (Stevenson et al., 1981).

Ovariectomized cows restricted in energy intake such that they
lost body weight responded to GnRH challenge with higher serum LH
concentration than ovariectomized cows that were gaining weight.
Depressed circulating LH concentration is not due to decreased
sensitivity of the anterior pituitary to GnRH (Beal et al., 1978).
Rutter and Randel (1984) found a trend toward higher GnRH induced LH
release in intact cows that maintained body condition compared with
intact cows that lost body condition postpartum. The apparent
discrepancy in LH response to GnRH between these two studies may be
explained by presence or absence of ovarian steroids.

The specific mechanism(s) responsible for depressed LH secretion
during restricted feeding is not clear. Recent work has demonstrated

that starvation—induced suppression of serum LH can be reversed by

 

33
administration of an opioid antagonist, naltrexone (Briski et al.,

1984). Possibly, restricted feeding suppresses circulating LH by
stimulating release of endogenous opioids from the hypothalamus which
inhibit LH secretion from the anterior pituitary. Campbell et al.
(1977) concluded that decreased release of gonadotropins during
starvation is due to reduced hypothalamic stimulation of the anterior
pituitary rather than the inability of the pituitary to secrete
hormones. Pulsatile secretion of LH seems to be an important factor
in stimulating follicular development and estrus expression (Haresign
et al., 1983). Increased frequency of LH pulses occur at the onset of
puberty, during the follicular phase of the estrous cycle and during
transition from periods of anestrous to estrous conditions (Haresign
et al., 1983). It would seem that increased frequency of LH pulses is
a prerequisite for resumption of estrous cycles.

Frequency of LH pulses in ovariectomized, restricted—fed heifers
was less than that of ovariectomized, adequately-fed heifers (Imakawa
et al., 1987). These authors concluded that feed restriction has a
direct action on the hypothalamic—pituitary axis to decrease frequency
of LH pulses. Restricting food intake of young female rats such that
weight gain was halted resulted in elimination of pulsatile LH
secretion (Bronson, 1987). Refeeding returned LH pulses within 48
hours. Furthermore, food restriction did not affect hypothalamic LHRH
content, LH content of the pituitary or response of the pituitary to
GnRH challenge. These results further support the suggestion that
restricted intake of feed directly suppresses LHRH release from the

hypothalamus.

 

34
Metabolic effects of restricted feeding in sows.

The frequent observation that prolonged intervals to estrus are
associated with excessive loss of body weight indicates that sows
were underfed relative to their nutritional requirements. Therefore,
restricted feeding with accompanying delayed return to estrus can be
used to study prolonged intervals to estrus. Many researchers have
conducted experiments which demonstrate the relationships among intake
of dietary essentials, changes in body weight and body condition and
interval from weaning to estrus. These studies were discussed earlier
in this review. Unfortunately, very few investigators have undertaken
controlled experiments which explain the mechanism by which these
clinical observations are related. The metabolic adjustments made by
a sow that is fed below nutritional requirements most certainly plays
a role in controlling the postweaning interval to estrus.

A reduction in dietary energy and protein of 90 and 67%
respectively, increased free fatty acid concentrations in serum but
did not consistently affect plasma insulin, glucose and urea nitrogen
compared with full-fed controls (Armstrong and Britt, 1987).

Armstrong et al. (1986) restricted dietary energy intake to 67% of ad
libitum intake for primiparous sows during lactation. There were no
consistent differences between feeding levels for preprandial serum
concentrations of insulin, glucose, free fatty acid or urea nitrogen.
Lack of a difference in blood borne metabolites may be explained by
the greater daily protein intake of restricted fed sows. But, sows
which displayed a short (< 8 d; estrous) or extended (> 23 d;
anestrous) interval to estrus ingested similar quantities of energy

and protein. No differences in body weight or backfat losses, litter

 

 

 

 

35
size at weaning or litter weight gain were observed for estrous and
anestrous sows. But, preprandial plasma glucose concentration was
higher and free fatty acid concentration was lower on days 12 and 20
of lactation for anestrous compared with estrous sows. From these

data, the authors concluded that some aberration in energy metabolism

 

which prevented a shift to spare glucose during lactation predisposed
sows to the occurrence of delayed postweaning estrus.

Cyclic sows exhibited greater frequency of LH release during the
last 18 h of lactation compared with anestrous females (Armstrong et
al., 1986). Similarly, Shaw and Foxcroft (1985) reported a negative
relationship between LH concentration before weaning and interval to
estrus. The ratio of FSH to LH was related positively to weaning to
estrus interval (Shaw and Foxcroft, 1985). FSH rises during late
lactation (Stevenson et al., 1981) and stays constant or rises slowly
during the postweaning period (Aherne et al., 1976; Shaw and Foxcroft,
1985). Therefore, a decrease in FSH/LH ratio which would be
associated with a shortened interval to estrus is due mainly to an
increase in LH concentration.

Low level feeding during lactation depressed postweaning LH
concentration in blood and extended interval to mating (Kirkwood et
al., 1987). No difference in LH release elicited by exogenous GnRH
could be detected when restricted-fed and full—fed sows were compared.
From this experiment one may conclude that the anterior pituitary of
nutritionally anestrous sows is responsive to hypothalamic
stimulation.

Armstrong and Britt (1987) reported a series of experiments in

 

 

 

36

which gilts were made anestrus by severe feed restriction.
Restricted-fed gilts had lower insulin and higher free fatty acid
concentrations in blood while glucose and urea nitrogen concentrations
were not different from full- fed controls. Mean LH and FSH
concentrations were not affected by feeding level but frequency of LH
release was lower and amplitude of release was higher in restricted-
fed gilts compared with full-fed gilts. In both groups, FSH secretion
showed no episodic release. Release of LH and FSH in response to
exogenous GnRH was not affected by feeding level indicating that the
anterior pituitary of restricted-fed gilts was responsive to
hypothalamic stimulation. Upon realimentation, LH pulse frequency
increased in restricted-fed gilts. In a second experiment,

nutritionally anestrous gilts received hourly intravenous pulses of

 

saline, GnRH or LH for 144 h. GnRH or LH decreased, six—fold, the
number of small follicles (1 to 3 mm) and increased ten-fold, the
number of large follicles (>7 mm diameter) indicating that ovaries of
nutritionally anestrous gilts are responsive to stimulation.

Summary.

Restricted feeding results in cessation of estrous cycles in
postpubertal females or prolongs anestrus postpartum. Furthermore,
restricted feeding is associated with depressed LH secretion while
resumption of estrous cycles is related to increased frequency of LH
secretion. Researchers have tested the anterior pituitary and ovaries
of nutritionally anestrous sows and gilts 1p yiyp and found them to be
functional and responsive to their appropriate hormonal stimuli. This

would suggest a lack of LHRH release from the hypothalamus associated

 

with restricted feeding is a major cause of nutritionally induced

 

 

 

anestrus in swine.

Restricted feeding causes catabolism of adipose and muscle
tissue. Catabolism of body tissue is related intimately to changes in
body metabolism. Considering this close interrelationship among
restricted nutrient intake, loss of body tissue and alterations in
body metabolism, the once simple model of anestrus in livestock
becomes increasingly complex (Figure 3). Low intake of dietary
nutrients results in simultaneous loss of body tissue and alterations
in body metabolism. Loss of body tissue may act directly to suppress
estrus or may act through a metabolite, hormone or catabolic
end product to suppress activity of HPO axis and suppress estrus.
Alternatively, loss of body tissue may be a tangential change
resulting from reduced nutrient intake and have no effect on estrus
expression.

On the other hand, restricted nutrient intake could affect the
hormonal and metabolic milieu of the body directly resulting in
suppressed activity of the HPO axis and anestrus. Although restricted
feeding suppressed all levels of the HPO axis, most evidence is that
central inhibition of hypothalamic LHRH release is a major cause of
nutritionally induced anestrus. The next obvious question is what
facet(s) of the hormonal and metabolic milieu associated with
restricted feeding is responsible for inhibition of activity of the
HPO axis? More specifically, what messenger mediates the effects of

restricted feeding on cessation of estrous cycles?

 

 

% _

38

 

 

relative to reproductive needs

/ \_

?
Loss of body :29 Hormonal and
tissue metabolic mediators

?
‘ Low Low
( Hypothalamus -———-> Anterior —-———+-
LHBH pituitary LH.FSH
\ _

Unlikely
\ pathway

Pow intake of dietary essentials]

 

 

 

 

 

 

    

 

Lovv
estroge

Clinical
observation

High incidence of anestrus l.-——__I

 

 

 

Figure 3. Relationships among low intake of dietary essentials,
body metabolism, change in body composition and incidence of
anestrus.

 

 

 

'39

Effect of Insulin on Reproductive Function

Insulin is a peptide hormone of central importance to
homeostatic regulation of metabolism in mammalian organisms. Insulin
has a wide variety of metabolic effects which include but are not

limited to enhanced glucose and amino acid transport, decreased

 

mobilization of adipose tissue, increased protein synthesis and
inhibition of protein degradation (Etherton, 1982). One of insulin’s
first recognized and primary roles is control of the movement of
metabolic fuels, primarily glucose. In the "fed" state, blood glucose
concentration rises stimulating release of insulin from the pancreas I
to facilitate transport of glucose out of blood and into body tissues.
Conversely, low blood glucose concentration characteristic of fasted
animals results in depression of serum insulin concentration.
Consequently, high levels of circulating insulin signal the "fed"
state (Cahill, 1971).
Since insulin has many actions and plays a primary role in
controlling metabolism, it is reasonable to speculate that insulin may
play a part in controlling reproductive processes. In previous
sections of this review, the positive relationship between restriction
of dietary nutrient intake and the occurrence of anestrus has been
established. Furthermore, under fasting conditions, there is a
consistent depression in insulin concentration in blood (Cahill et
al., 1966; Cahill, 1971; Kasuga et al., 1977; Wangsness et al., 1981;
Armstrong and Britt, 1987). In a cursory analysis of these
observations, one may hypothesize that the occurrence of anestrus

caused by depressed nutrient intake may be mediated through depressed

 

4O

systemic insulin concentration. In a more detailed investigation of
the relationship between insulin and reproductive function one finds
several lines of evidence that suggest insulin can influence
reproductive processes.

Diabetes as a model for effects of insulin on reproduction.

Juvenile onset diabetes is characterized by impaired release of
insulin from the pancreas in response to blood glucose. This
aberration in carbohydrate metabolism results in hypoinsulinemia,
hyperglucosemia and hyperglucosuria (Mayes, 1983). In addition,
insulinopenia has been associated with primary amenorrhea, anovulation
and low pregnancy rate in women (Poretsky and Kalin, 1987) and
prolonged estrous cycles, anestrus and anovulation in rats (Liu et
al., 1972; Kirchick et al., 1978). So, it seems that diabetes would
be a useful model for investigating the relationship between insulin
and reproductive function.

Alloxan or streptozotocin are used routinely to create a diabetic
state in experimental animals (Anonymous, 1976). Both act by
destroying insulin-releasing B cells of the pancreas. Immature
alloxan diabetic rats experienced ovarian and uterine atrophy and had
elevated serum FSH concentrations but normal LH profiles compared with
normal controls. None of the alloxan treated rats experienced vaginal
opening. Insulin administration to alloxan diabetic rats increased
ovarian and uterine weights, depressed FSH to normal concentration and
caused vaginal opening in 90% of the rats (Liu et al., 1972).

In contrast, there was no difference in ovarian weight or

estradiol production between diabetic and normal immature rats that

 

 

 

41

were primed with PMSG (Kirchick et al., 1978). This suggests that
ovarian responsiveness to gonadotrophic stimulation is not depressed
in diabetic animals. Treatment of diabetic rats with insulin or
human chorionic gonadotropin resulted in ovulation in 90% of the
animals. Non-ovulatory diabetic rats displayed no LH surge (Kirchick
et al., 1978). This indicates that the pituitary and(or) hypothalamus
are limiting reproductive function in diabetic animals.

Depletion of hypothalamic LHRH determined by serial slaughter
technique was similar between diabetic and normal rats after time of
expected LH surge. Similarly, hypothalamic content of LHRH was not
different between the two groups (Kirchick et al., 1979). A single
injection of GnRH resulted in a three- to four-fold greater release of
LH in control rats than diabetic rats. Furthermore, exogenous GnRH
did not reduce LH concentration of pituitary in diabetic rats
(Kirchick et al., 1979). Evidently, the pituitary is not responsive
to GnRH stimulation in the diabetic rat. Depressed responsiveness of
the pituitary seems to be insulin—dependent because insulin
replacement in diabetic rats re-established the preovulatory surge of
LH (Kirchick et al., 1978) and ovulation (Kirchick et al., 1982) in
immature rats. Insulin treatment of diabetic rats prevents
hypothalamic lesions and atrophy of the pituitary normally observed in
diabetic rats (Bestetti et al., 1987).

In a recent review, Poretsky and Kalin (1987) discussed the
effects of diabetes on human reproduction. Normal ovarian activity
returns with administration of insulin to diabetic women. They
suggested that insulin has a gonadotrophic effect. However, the

authors warned that id vivo effects of insulin in humans must be

 

 

42

interpreted cautiously because one cannot determine which metabolic
aberration of uncontrolled diabetes causes the abnormalities in
ovarian function. Hyperglycemia, insufficient body weight, insulin
deficiency, gonadotropin deficiency or any combination of these
factors could disrupt normal ovarian function.

In vitro effects of insulin on HPO axis.

 

Since there are numerous potentially confounding factors present

when studying 1p vivo effects of insulin, effects of insulin in vitro

 

have been examined extensively. I vitro effects of insulin have been

 

demonstrated at all three endocrine glands of the HPO axis.

Insulin has direct effects on ovarian tissues. Specific binding
sites for insulin have been demonstrated on porcine granulosal cells
(Rein and Schomberg, 1982; Otani et al., 1985). Hammond and English
(1987) showed that insulin is a potent stimulator of DNA synthesis in
porcine granulosal cells. Insulin prevented decline in basal
secretion of progesterone normally observed in granulosal cells (May
and Schomberg, 1981). Furthermore, maturation of immature granulosal
cells (May and Schomberg, 1981) and luteinization of granulosal cells
(Channing et al., 1976; Otani et al., 1985) is stimulated by insulin
in culture media.

Follicle stimulating hormone caused a dose related increase in
FSH binding to granulosal cells only when insulin was present in the
culture medium (May et al., 1980). Insulin is required for
development of optimal steroidogenic potential of granulosal cells
(May et al., 1980; Lino et al., 1985) and acts synergistically with

FSH (Maruo et al., 1988). A major function of granulosal cells is

 

 

 

 

43

steroidogenesis during follicular phase of the estrous cycle. Insulin
synergizes with gonadotropins to promoting steroidogenesis.

In addition to ovaries, insulin has direct effects on the
pituitary. Receptors for insulin have been demonstrated in the
pituitary (Havrankova et al., 1978a). Anterior pituitary cells from
ovariectomized adult rats responded to insulin treatment with
increased basal and GnRH stimulated release of LH and FSH (Adashi et
al., 1981).

The blood—brain barrier is a mechanism by which the brain is
protected from radical shifts in body metabolism. It was believed
that the blood-brain barrier was impermeable to insulin. However,
recent experiments have shown that insulin rapidly enters the
cerebrospinal fluid and enters neural tissue (Baskin et al., 1987).
Havrankova et al. (1978b) reported that the hypothalamus of rat brain
had the highest concentration of insulin among six regions of the
brain. Receptors for insulin have been demonstrated in the
hypothalamus (van Houten et al., 1979; Landau et al., 1983). Access
of insulin to the hypothalamus may provide direct interaction between
the control centers of the brain and body metabolism (van Houten et
al., 1983; Melnyk and Martin, 1984a).

Recent evidence indicates that plasma insulin acts as a satiety
signal to control feed intake and body weight (Woods et al., 1985)
thus supporting the role of insulin as a messenger between body
metabolism and the brain. Prolonged food restriction did not affect
hypothalamic insulin concentration but reduced binding of insulin to
receptors in rat hypothalami (Melnyk and Martin, 1984a). Presumably,

reduced binding of insulin to receptors decreases release of LHRH. If

 

 

 

 

 

44

this is true, one may hypothesize that effects of feed restriction
resulting in anestrus are mediated by reduced insulin binding in
hypothalamus. Apparently, there is maximal binding of insulin in
hypothalamus because hyperinsulinemia did not increase hypothalamic
binding of insulin (Melnyk and Martin, 1984b).

Effects of insulin on estrous cycles of farm animals.

The relationship among insulin, HPO axis and expression of estrus
is a new area of investigation in farm animals. Relatively few 1p
yiyd studies have been reported. Fasted heifers had lower plasma
insulin and glucose concentrations compared with ad libitum-fed
heifers (McCann and Hansel, 1986). Associated with depressed insulin
and glucose concentrations was decreased mean concentration of LH in
plasma. Upon refeeding of fasted heifers, increased plasma insulin
concentration preceeded increased LH concentration. In sows with
short intervals from weaning to estrus, terminating lactation by
weaning of pigs results in a rapid rise in insulin while glucose
concentrations were unchanged during the first four days postweaning
(Eriksson et al., 1987). Concurrently, there is an increase in mean
concentration of gonadotropins, pulse frequency of LH, and follicular
growth which ultimately results in behavioral estrus and ovulation
(Stevenson et al., 1981; Shaw and Foxcroft, 1985). These temporal
relationships between insulin and LH secretion indicate that insulin
may stimulate the hypothalamic-pituitary axis to resume normal
activity.

If insulin does have a role in stimulating activity of the

hypothalamic-pituitary axis then administration of insulin to

 

 

 

 

 

 

 

 

45
restricted-fed females may increase secretion of gonadotropins and
restore expression of estrus. Short term (4h) infusion of insulin did
not affect pattern of LH release in ovariectomized heifers (Harrison
and Randel, 1985). Restricting energy intake of heifers for 45 d
caused weight loss, decreased postprandial insulin concentration and
decreased ovulation rate (Harrison and Randel, 1986). Daily
injections of insulin had no effect on mean LH concentration or
patterns of LH secretion; however, exogenous insulin increased
ovulation rate and secretion of progesterone in restricted-fed
heifers. Evidently, exogenous insulin mimicked the metabolic state of
adequately-fed heifers and restored ovulation rate of restricted-fed
heifers to near normal levels. Lack of an effect of insulin on LH
secretion would indicate that effects of insulin are localized at the
ovaries. Similar to effects in heifers, Cox et al. (1987) reported
that exogenous insulin increased ovulation rate of gilts consuming
moderate and high energy rations. Effect of insulin on ovulation
rate in gilts consuming moderate energy rations was greater than in
gilts consuming high energy rations. Increasing ovulation rate due to
administration of insulin occurred independent of changes in pulsatile
release of LH.

Presently, it is not possible to determine if 1p yiyd effects of
insulin are direct or indirect resulting from insulin-elicited changes
in concentration of glucose. Some researchers have pr0posed that
glucose concentration in plasma rather than insulin may be the signal
which stimulates the HPO axis (Short and Adams, 1988). Phlorizin-
induced hypoglycemia decreased LH pulse amplitude and the number of

medium amplitude (>2 ng/ml) LH pulses (Rutter and Manns, 1988).

 

 

 

 

 

 

 

 

 

46

Despite changes in LH release, phlorizin did not affect the number of
cows ovulating within 24 days of treatment (Rutter and Manns, 1987).
But, phlorizin-induced hypoglycemia was confounded by hypoinsulinemia.
A metabolic inhibitor of glucose metabolism (2-deoxy-D-glucose)
blocked estrus and ovulation in beef cows (McClure et al., 1978)
suggesting that glucose independent of insulin is necessary for
expression of estrus.

A goal of this dissertation is to elucidate in part the factors
responsible for anestrus in highly productive sows. There have not
been comprehensive studies which directly compare the metabolic status
of sows with short or extended intervals to estrus. Consequently, it
is not known if anestrous sows have depressed concentrations of
insulin in plasma compared with regularly cycling sows. However,
based on the preponderance of evidence that insulin does influence
function of the HPO axis, a hypothesis to test is that insulin may
affect estrus expression positively in nutritionally anestrous sows.
Summary.

Exogenous insulin in diabetic animals normalizes aberrant
functions of the HPO axis and elicits normal estrous cycles. From ip
yitpd observations, insulin influences positively function of the
hypothalamus, pituitary and ovaries. Exogenous insulin increased
ovulation rate in heifers and gilts. From this evidence, one may
speculate that insulin has a positive effect on HPO axis and that

exogenous insulin may have beneficial effects on nutritionally induced

anestrous in sows.

 

 

 

 

 

 

 

 

 

47

mm

The relationship among low intake of dietary nutrients, body
composition and body metabolism and extended interval from weaning to
estrus is complex and can be visualized by referring to Figure 3. Low
intake of nutrients causes simultaneous changes in body composition
and body metabolism. Loss of body tissue below a minimal threshold of
specific tissues (e.g., fat or muscle) may cause anestrus directly.
Another possibility is that changes in body composition alter hormones
or metabolites which depress activity of the HPO axis and result in
anestrus. Clinical observations of women and farm livestock indicate

that body fat and occurrence of regular menstrual or estrous cycles

 

are related positively. But, this relationhip has not been tested
adequately. The first objective of this research was to determine if
a minimal threshold of body fat exists below which interval from
weaning to estrus is delayed in primiparous sows. A second objective
was to determine the relationship between body fat and postweaning
interval to estrus.

Independent of changes in body composition, low dietary intake of
nutrients could alter body metabolism such that normal functions of
the HPO axis are depressed resulting in anestrus. There are numerous
changes in body metabolism caused by restricted intake of dietary
essentials. Consequently, many different hormones or metabolites may
be responsible for nutritionally induced anestrus. Insulin is a
metabolic hormone of central importance in regulating homeostasis.
Insulin responds to intake of dietary essentials.

Previous researchers have established that insulin has positive

 

 

 

48

effects at all levels of the HPO axis. Therefore, insulin is a
hormone related to body metabolism which may influence expression of
estrus. Effects of exogenous insulin on interval from weaning to
estrus in sows have not been tested. The third objective of this

research was to determine effects of exogenous insulin on interval to

postweaning estrus in restricted-fed primiparous sows.

 

 

 

 

 

Experiment I
Relationship Between Body Fat and Postweaning Interval to Estrus in

Primiparous Sows

49

 

50

Introduction

—I-

Extended intervals from weaning to estrus (210 d) are common n
highly productive maternal—line sows. Delayed postweaning estrus and
associated delayed rebreeding of sows decreases reproductive

efficiency and increases costs of production. MacLean (1968, 1969)

 

observed that in lean sows delayed postweaning estrus occurred more
frequently than in moderately fat sows. Large losses of body weight
and fat during lactation were correlated positively with delayed
postweaning estrus (Reese et al., 1982; King and Williams, 1984; King
and Dunkin, 1986). Similarly, postpartum beef cows that lost body
weight during lactation displayed estrus later than cows that did not
lose body weight (Cantrell et al., 1982; Rutter and Randel, 1984).

The percentage of dairy cows that displayed estrus by d 24 postpartum

 

increased linearly as body condition improved (Fulkerson, 1984).

Previous researchers have hypothesized that a minimal threshold
of body fat is necessary in women for menarche and sustained menstrual
cycles (Frisch and McArthur, 1974; Frisch, 1984). Several workers
have reported irregularity and(or) cessation of menstrual cycles in
female athletes with lower body fat than that of regularly cycling
women (Dale et al., 1979; Carlberg et al., 1983; Glass et al., 1987).
In contrast, Sanborn et al. (1987) found no relationship between body
fat and amenorrhea in women athletes.

The objective of this experiment was to determine if a minimal
threshold of body fat exists below which postweaning estrus is
delayed. A second objective was to examine the relationship between

body fat and interval from weaning to estrus in primiparous sows.

51
Materials and Methods

Animals and Management. On d 109 of gestation, twenty-two
primiparous Yorkshire sows were moved to farrowing crates in an
environmentally controlled room. From d 109 of gestation until
parturition, all sows received 1.82 kg/d of a corn—soybean meal
gestation diet (Table 1). Sows were allotted, based on weight at d
109 of gestation and ancestry, to receive one of four diets starting
at parturition (Table 1). Diets were formulated and feed intake of
lactating sows was controlled to provide 7 (VL), 9 (L), 11 (M) or 13
(H) Mcal of metabolizable energy (ME) daily to produce different
degrees of body fatness at weaning. The diet which provided 13 Mcal
ME was considered marginally adequate in energy for primiparous sows
nursing ten pigs. The remaining diets (VL, L, M) represented varying
degrees of energy restriction. These diets were used to produce thin
sows with extended intervals to postweaning estrus. Calculated intake
of all dietary essentials except ME met or exceeded NRC (1979)
recommendations for lactating sows consuming 4 kg of feed daily (Table
2).

During lactation, sows received half of their daily feed
allotment at 0730 h and at 1530 h. Orts were weighed and recorded
daily for each sow to determine daily intake of dietary essentials.
Litter size for all sows was adjusted to ten pigs by d 3 postpartum.
Body weights of sows were recorded on d 109 of gestation, 24 h
postpartum and 28 d postpartum (weaning). In addition, backfat depth
was determined ultrasonically5 at a point 60 mm lateral to the dorsal
midline opposite the last rib on d 109 of gestation and at weaning.

Individual pig weights were recorded at 24 h and 21 d postpartum.

 

 

 

52

TABLE 1. COMPOSITION AND CALCULATED ANALYSIS OF DIETS

 

 

 

 

 

 

 

 

 

PDiets
Ingredient Very Low Low Medium High Gestation
(VL) (L) (M) (H)
................... %---------------_---_---
Corn 44.35 56.35 67.20 62.05 70.45
Soybean meal 38.60 26.60 24.50 23.50 14.50
Wheat bran 10.00
Solka gioca 11.15 12.25
Tallow 3 50 10.00
Mono—dicalcium phosphate 2.30 1.75 1 70 1.50 2 00
Calcium carbonate 1.75 1.50 1.55 1.40 1.30
Salt .60 .50 .50 .50 .50
Vit.-trace mineral gixC .60 .50 .50 .50 .60
Vit. E-selenium mix .60 .50 .50 .50 50
Aureomycin-Soe .05 .05 .05 .05
Choline chloride, 50% ' .15
100 00 100 00 100.00 100 00 100 00

 

Calculated analysis
Metabolizable energy

(ME), kcal/kg 2612 2654 3243 3578 2996
Crude protein, % 20.8 16.6 16.6 15.8 14.0
Lysine, % 1.22 .90 .87 .82 .64
Calcium, % 1.23 1.00 1.00 .91 .92
Phosphorus, % .86 .70 .69 .64 .80

 

dPowdered cellulose; James River Corporation, Berlin, NH 03570.

bRENDO White Beef fat; Michigan Shortening Co., Detroit, MI 48207.
CComposition was vitamin A, 661,380 IU; vitamin D , 132,276 1U;
menadione, .66 g; riboflavin, .66 g; niacin, 3.5 g; D-pantothenic
acid, 2.64 g; choline 88.18 g; vitamin 812, 3.96 mg; Zn, 7.5 9; Mn,
7.5 g; I, .11 9; Cu, 2.0 9; Fe, 12.0 g/kg premix.

Composition was vitamin E, 3310 IU/kg premix and selenium, 19.8
mg/kg premix.

eProvided 110.23 9 chlortetracycline per kg premix.

d

53

 

 

 

TABLE 2. DAILY ALLOWANCE 0F FEED AND SELECTEDa NUTRIENTS FOR
PRIMIPAROUS SOWS DURING LACTATIONa
Dietary energy levels

Nutrient VL L M H
Total feed, kg/g 2.73 3.41 3.41 3.64
Energy, Mcal ME /d 7.11 9.03 11.03 12.98
Crude protein, g/d 568 568 568 573
Lysine, g/d 33.2 30.7 29.5 29.9
Calcium, g/d 33.6 34.0 34.4 33.0
Phosphorus, g/d 23.3 23.7 23.5 23. 3

 

dCalculated values.
Metabolizable energy.

 

54
Pigs did not receive creep feed.
Beginning one day after weaning, all sows received gestation diet
(Table 1) in an amount based on their metabolic body weight (kg
BW'75). Each sow received .037 kg feed (110 kcal ME)/kg BW'75/d to

satisfy maintenance energy requirements plus an additional .45 kg feed

 

(1,359 kcal ME) for body weight gain. The amount of feed offered was
adjusted each day based on sow weight. From d 3 to d 65 postweaning,
sows were checked daily for signs of estrus using a mature boar.
Estrus was recorded when sows stood to be mounted by a boar and days
from weaning to first estrus were recorded. Blood was sampled via
puncture of the vena cava (Carle and Dewhirst, 1942) from all sows
that did not exhibit estrus by 14 d postweaning. Subsequent blood
samples were obtained biweekly until estrus was detected. Plasma was
harvested by centrifugation at 5°C and stored at —20°C until assayed
for progesterone.

Progesterone Assay. The radioimmunoassay for progesterone (Louis
et al., 1973) was validated for porcine plasma. First, extraction
efficiency at various volumes of plasma was determined. Ten
microliters of tritiated progesterone were added to 16 x 100 mm
extraction tubes and the methanol diluent evaporated. To each tube,
different volumes of plasma (100, 200, 500 or 1000 ul) were added and
vortexed for 10 s. Progesterone was extracted using 2 ml of fresh
toluene:hexane (1:2) and 30 s of vortexing. Mean efficiency of
extraction declined as the volume of plasma increased. Maximal
extraction efficiency (92%) was achieved with 100 ul of plasma.

Extraction efficiency at various concentrations of progesterone was

 

 

 

55

determined by adding 19,500, 28,800 or 38,200 cpm of tritiated

 

progesterone to 100 ul of plasma. Extraction proceeded as described
previously. The percentage of progesterone recovered was constant
regardless of the amount of progesterone present.

Accuracy was determined by adding 0, .25, .50 or 1.0 ng
progesterone to 100 ul of plasma. Each sample was extracted as
described previously and assayed by the procedure of Louis et al.
(1973). After correction for endogenous progesterone in plasma,
content of progesterone measured in each tube did not differ from
exogenous progesterone. Intra-assay coefficient of variation was 7.0%
for both high and low standard sera. Concentration of progesterone in .
the standard high and low sera was 2.32 i .16 and .25 i .02 ng/ml,
respectively. There was no inter-assay coefficient of variation
because all samples were analyzed in one assay. Presence of
functional corpora lutea was assumed when plasma progesterone
concentration was > 1 ng/ml. Since blood was sampled only from sows
not detected in estrus, elevated plasma progesterone would have
indicated ovulation in the absence of behavioral estrus.

Body Fat. 0n the day before weaning and the day of first
postweaning estrus, body fat of sows was estimated by dilution of
deuterium oxide (D20). Each sow received .24 g of D201 per kg of body
weight. Sodium chloride was added to pure D20 to create a
physiological saline solution (.85% NaCl). Exact amount of
physiological DZO injected was determined from the pre- and post-
injection syringe weight. Injection of D20 was via polyethylene

m

199.8% pure DZO obtained from Cambridge Isotope Lab, Woburn, MA.

56
catheter in the anterior vena cava. Catheters were flushed
immediately with 10 ml of heparinized saline to assure that all 020
was in the vascular system. Blood was sampled (10 ml) in EDTA-treated
syringes before D20 infusion and 2 and 3 h after infusion of 020.
Blood was stored at 2°C until completion of sampling for a day, then
all samples were frozen in sealed tubes and stored at -20°C until
recovery of blood water.

Blood samples were lyophilized according to the procedures of
Byers (1979). Water extracted from blood was stored in sealed tubes
at 2°C until quantification of 020 by infrared absorbance2 (Byers,
1979).

Estimates of maternal body (ingesta-free sow body minus uterus
and mammary tissue and their contents) fat at weaning were calculated
using equations of Shields et al. (1984). Empty body (ingesta—free
sow body) fat content at first postweaning estrus was estimated using
equations of Knudson et al. (1985).

Statistical Analysis. A hypothesis is that low body fat at
weaning may extend the anovulatory interval. To test this hypothesis,
regression analyses (first and second order) were used to examine the
relationship between body fat, the independent variable, and interval
from weaning to estrus (Gill, 1978). Based on previous reports (Reese
et al., 1982; Nelssen et al., 1985), sows fed M and H diets were

expected to have short weaning to estrus intervals. Therefore, fewer

 

2Infrared absorbance measured by a Wilks Scientific Miran 1 Fixed
Filter Infrared Analyzer with a Gilford Model 410 digital unit.

 

57
sows were assigned to M (n=4) and H (n=2) than to VL (n=8) and L
(n=8). This unbalanced approach was planned to improve the estimate
of the interval from weaning to estrus of lean sows. Since
replication was not equal, simple linear regression was used (Gill,
1978) to analyze the relationship of dietary ME allowance with each of
the following dependent variables: daily ME intake, body weight and
backfat depth of sows, litter performance and interval from weaning to
estrus. Student’s t statistic was used to contrast the performance of
sows that displayed short (5 10 d) and long (> 10 d) intervals to

estrus.

Results

As expected, a positive linear effect (P<.01) of dietary ME
allowance on average daily ME intake was observed (Table 3). At 24 h
postpartum, body weight was not different among diets. But, body
weight of sows at 28 d postpartum increased linearly (P<.05) with
amount of dietary ME offered. Backfat depth 24 h postpartum declined
linearly (P<.05) as daily ME allowance increased. In contrast,
backfat depth at weaning was not affected by ME allowance. Dietary
energy allowance did not affect litter size on d 21 of lactation.
Similarly, litter weights on d 21 of lactation were not different
among dietary energy levels.

Loss of body weight and backfat during lactation was related
inversely (P<.01) to ME intake (Table 4). Increasing ME intake during
lactation increased (P<.01) body fat at weaning and was related
negatively (P<.OI) to postweaning interval to estrus.

Body fat at weaning ranged from 14.5 to 30.8% (Figure 4). There

 

 

 

-L_

 

58

TABLE 3. EFFECT OF METABOLIZABLE ENERGY INTAKE 0N SON WEIGHT,
BACKFAT DEPTH AND PIG PERFORMANCE

 

 

Trait VL L M H Sy’xa Pb
No. of observations 8 8 4 2
Avg energy intake,
Mcal ME /d 6.88 8.72 11.03 12.93 .26 .01
Sow wt, kg: d
24 h postpartum 168.6 175.5 173.4 180.1 21.1 NS
Weaning 131.1 140.6 143.9 159.9 21.7 .05

Sow backfat depth, mm:

24 h postpartum 23.3 23.18 21.5 18.1 2.95 .05
Weaning 11.3 13.7e 14.8 13.5 2.89 NS
21 d litter size 9.0 9.5 9.5 9.0 .9 NS
21 d litter wt, kg 48.9 51.6 47.4 47.8 9.9 NS

 

EStandard error of the estimate.
Probability level.
gMetabolizable energy.

Not significant (P>.05).

Means represent 7 observations.

 

59

TABLE 4. EFFECT OF DIETARY ENERGY INTAKE DURING LACTATION ON SON
BODY FAT AND NEANING TO ESTRUS INTERVAL

 

Dietary energy levels

 

Trait VL L

M H Sy'xa Pb

 

No. of observations 8 8

Avg energy intake,
Mcal ME /d

6.88 8.72
Lactational wt loss, kg 37.5 34.9
Lactational backfat loss, mm 12.0 9.4d

Maternal body fat at
weaning, 2.9 18.3 21.4

Weaning to estrus interval, d 30.1 12.9

11.03 12.93 .26 .01
29.5 20.2 11.0 .01
6.8 4.5 3.0 .01

25.9 30.8 3.4 .01
5.2 4.5 11.6 .01

 

“Standard error of the estimate.
Probability level.
CMetabolizable energy.

Mean represents 7 observations.
eMeans represent 7, 7, 3 and 1 observations
levels, respectively.

 

for VL, L, M and H energy

L

 

 

 

 

 

I w

60— O

55—-

50—-

45 - o Y=52.5l- l.59X

4O __ r2=o.24
(p<.05)

35~ 0
so—
25»-
20-
is»

Postweoning lniervol to Estrus (d)

5r-

_10221 I_ l I I I I I I
I4 18 22 26 3O

Moternol Body Fot oi Weoning (°/o)

 

 

Figure 4. Relationship of maternal body fat of primiparous sows at
weaning to interval from weaning to estrus. Data from four sows
were deleted from the analysis due to technical problems with
D20 dilutions (n=18).

 

 

 

 

 

 

 

 

61

was a linear (r2=.24; P<.05) association between body fat at weaning
and interval from weaning to estrus. Body fat at estrus ranged from
26.6 to 36.0%, but there were no significant linear (r2=.03) or
quadratic (r2=.03) relationships between body fat at first postweaning
estrus and interval from weaning to estrus (Figure 5). Percentage of
backfat present at 24 h postpartum that was lost during lactation
ranged from 16.6 to 66.6% (Figure 6). A linear (r2=.24; P<.05)
relationship between relative backfat loss during lactation and
weaning to estrus interval was observed.

Independent of body fat, sows either displayed estrus by d 10
postweaning or resumption of ovarian activity was delayed until after
d 21. Thus, distribution of postweaning estrus was bimodal as
reported previously (Armstrong et al., 1986; Johnston et al., 1987).
Low dietary energy intake during lactation was associated with an
extended interval to postweaning estrus (Table 5). Furthermore, sows
with delayed return to estrus experienced greater loss of weight and
backfat (P<.05) compared with sows that exhibited estrus promptly.
Litter weight at d 21 of lactation did not differ between sows that
displayed short or extended intervals from weaning to estrus.

Percentage of body weight at 24 h postpartum lost during
lactation ranged from 10.8 to 30.6% (Figure 7). Percentage loss of
body weight explained a greater proportion of the variation (r2=.32;
P<.05) in interval to estrus than any measure of body fat (Figure 4,
5). Metabolizable energy intake during lactation accounted for the

largest portion of the variation (r2=.48; P<.01) in interval from

weaning to estrus (Figure 8).

 

 

 

 

62

 

60r O

55—-
3 50L—
U? - _.
2 45__ Y-44.55 0.92x
; r2=oos
“é 40" (p>.50)
'3: 35—
9
3:3 30-
C:
'— 25"“ CD 0

c»

S? 20.. O O
S I.
G)
5 I5
m TOF-
a a o o O

5“ o 000

l l l L l l

 

 

—-//
O 26 28 30 32 34 36

Empty Body Fat oi Estrus (°/o)

Figure 5. Relationship of empty body fat of primiparous sows at first
postweaning estrus to interval from weaning to estrus. Data from
five sows were deleted from the analysis due to technical problems

with 020 dilutions (n=17).

 

 

 

 

 

 

63
soI O
55—-
3 50—
U)
f; 45— - Y=-4.70+O.53X o
11"] 40- r2=o.24
+9 (p<.05)
[)3 35'— O
.32 soL /
.E
I? 25— O O o/0 o
5 20* O V
O)
E l5~
8
0. IO— 000 o o
0
50 o oo o
illliltltltllliltltlil

 

20 30 4O 50 60 70
Loss of Bockfoi During Locioiion (°/o)

Figure 6. Relationship between backfat loss during lactation
(percentage loss of 24 h postpartum backfat) and interval from
weaning to estrus (n=21).

 

 

64

TABLE 5. ENERGY INTAKE AND PERFORMANCE OF PRIMIPAROUS SONS THAT
DISPLAYED SHORT OR LONG INTERVALS TO ESTRUS

 

Interval to estrus

 

Item 5 10 d SEMd > 10 d SEM

 

No. of observations 12 10

Lactational energy intake, Mcal MEb/dC 10.08 .54 7.39 .28

Lactational wt loss, kgd 29.8 2.8 38.0 1.3
Relative wt loss, %de 17.6 1.7 21.9 .9
Lactational backfat loss, mmd 7.7 1.0 11.3 1.2
Relative backfat loss, %df 35.5 3.8 47.7 4.3
Litter wt d 21 of lactation, kg 49.2 2.2 49.9 1.7

 

“Standard error of the mean.

Metabolizable energy.
CMeans within a row differ (P<.01).

Means within a row differ (P<.05).
ePercentage of 24 h postpartum wt lost during lactation. .
Percentage of 24 h postpartum backfat depth lost during lactation.

 

65

 

GOI' O
55—
% Y=——63.59+7'.45x—o.lsx2
v so~ 2
U) r =O.3O
53 45 - (p<.05) O
if)
m 40—
.9
a 35‘ o
i
f‘.’ .50—-
E 25— o o
(3‘
E 20 _ OO \
8 l5
5
‘” IO—
CE? (3 (JCIC) () C)
5 ”‘ 80’ Q)
C) 7//tl .//I I I l I I I I l

 

1
IO I4 I8 22 26 30

Relative Weight Loss
During Locioiion (°/o)

Figure 7. Relationship between relative weight loss during lactation
(percentage loss of 24 h postpartum weight) and interval from
weaning to estrus (n=22).

 

 

l

55

50 _. y:l43.l7—23.24x+o.97x2
r2=o48

45 _ O (p<.Ol)

4o —

  

Postweaning interval to) Estrus (d)

  
 

 

 

35? O

30—

25*

20-

15.-

lot-

0

5“ o ‘3)
O_//lllllilJ

67 8 9|OI|I2I3
Dolly ME lm‘oke During Lociotion (Mcol)

Figure 8. Relationship between daily metabolizable energy (ME) intake
during lactation and interval from weaning to estrus.

 

 

67

Discussion

A positive linear effect of dietary ME intake on body weight of
sows at weaning and a negative relationship between lactational energy
intake and loss of body weight were not unexpected. Similar decreases
in loss of body weight as daily intake of energy increased have been
reported (Reese et al., 1982; King and Williams, 1984; Nelssen et al.,
1985; Armstrong et al., 1986). In contrast, a linear decline in
backfat depth 24 h postpartum as lactational ME allowance increased
was unexpected. Dietary energy levels imposed at parturition would
not be expected to have a biological effect on backfat depth 24 h
postpartum. A more plausible explanation is that two rather lean sows
were assigned randomly to the highest level of dietary energy (H)
which resulted in a statistically significant difference among
lactational energy levels. Due to the lean sows assigned to the
highest energy level, backfat depth at weaning was not affected by
lactational energy intake.

Litter size and weight on d 21 of lactation were not influenced
by lactational energy allowance. One would not expect litter size to
be affected by energy allowance because all litters were adjusted to
ten pigs by d 3 postpartum. Restricting ME intake by primiparous sows
to 10 Mcal/d reduced litter weight at d 28 postpartum compared with
sows receiving 12 or 14 Mcal/d (Nelssen et al., 1985). Reese et al.
(1982) reported that restricted intake of ME by primiparous sows (8
Mcal/d) did not reduce pig weaning weight compared with litters from
sows receiving 16 Mcal ME/d. Evidently in the study reported herein,

sows receiving restricted amounts of ME mobilized enough body reserves

 

 

68

to satisfy the energy demands of lactation.

Body weight and backfat losses were related negatively to ME
intake during lactation. This negative relationship yielded a
positive relationship between ME intake and body fat at weaning.
There was an inverse relationship between body fat at weaning and the
interval from weaning to estrus. Short intervals from weaning to
estrus have been associated with minimal loss of body fat (Reese et
al., 1982; King and Williams, 1984b; Nelssen et al., 1985).
Similarly, MacLean (1968, 1969) reported that sows weaned in thin body
condition displayed extended intervals to first postweaning estrus.
But, in these and all previous studies and the analysis presented in
Table 4, dietary energy and body fat were confounded. Dietary energy
intake during lactation was manipulated to produce sows of different
body fatness at weaning. Therefore, one cannot determine if changes
in the interval to estrus were due to dietary energy intake or body
fat.

A hypothesis is that low body fat at weaning may extend the
anovulatory interval. To test this hypothesis, body fat and interval
to estrus were regressed without regard to intake of ME during
lactation. Creation of a prolonged interval from weaning to estrus
and sows with low fat were prerequisites for an appropriate test of
this hypothesis. Low dietary ME offered during lactation prolonged
interval to estrus and lowered body fat of sows. Relationship between
total body fat of sows and interval to estrus have not been reported
previously. Sows with less than 13.1% body fat as estimated from
carcass fat content displayed a delayed return to estrus. Percentage

body fat reported in the present study was higher than that reported

 

“MI

69
by Reese et al. (1984) probably because estimates of body fat using
D20 dilution accounted for internal body fat stores.

Although the coefficients of determination for relationships
between body fat and interval to estrus were significant (Figures 4,6)
body fat or backfat loss accounted for less than 25% of the variation
in interval from weaning to estrus (Figures 4, 5, 6). Low
coefficients of determination (<.25) suggest that body fat may not be
a primary controller of the interval from weaning to estrus. In
addition, one cannot predict with accuracy the weaning to estrus

interval using body fat of restricted-fed primiparous sows as the

 

independent variable. Neither backfat depth (average of 3 sites) nor
visually determined condition score measured during lactation were
correlated to the percentage of sows displaying estrus by d 12
postweaning (Esbenshade et al., 1986). Lack of a relationship between
body fatness and regularity of menstrual cycles in women has been
reported previously (Baker et al., 1981; Sanborn et al., 1987).

A bimodal distribution of postweaning interval to estrus has been
observed by others (Armstrong et al., 1986; Johnston et al., 1987).
Delayed postweaning estrus was not due to ovulation without detected
estrus since progesterone in plasma was below 1 ng/ml in all sows with
extended (>10 d) weaning to estrus intervals. It has been suggested
that negative energy balance may be an important causative factor of
amenorrhea in athletic women (Warren, 1980). One may hypothesize that
sows with a delayed return to estrus (>10 d) experienced greater
negative energy balance during lactation due to higher milk production

than sows that experienced a short interval to estrus. Milk

 

70

production was not measured directly. However, litter weight is
correlated positively with milk production (Ai et al., 1973; Lewis et
al., 1978) and can be used as an indicator of milk production. Litter
weight at d 21 of lactation did not differ for estrous and anestrous
sows. Similar weight gain of litters nursing sows that displayed

short or extended intervals from weaning to estrus has been reported

 

previously (Reese et al., 1984; Armstrong et al., 1986). Evidently,
volume of milk produced was similar between estrous and anestrous sows
which would suggest that energy demands of lactation were similar for
these two groups of sows. Sows with extended intervals to estrus lost
a greater percentage of weight and backfat during lactation than sows
with short intervals to estrus. Furthermore, daily ME intake during
lactation was lower for sows with a long interval to estrus than sows
that displayed a short weaning to estrus interval. Low daily intake
of ME during lactation forced sows to mobilize large quantities of
body tissue to meet energy demands of milk production. Ultimately,
energy deficit during lactation resulted in delayed return to
postweaning estrus.

Loss of body weight during lactation explained more of the
variation in interval to estrus than either measure of body
fat or loss of backfat. Sows that lost the most weight during
lactation had extended anovulatory intervals (Reese et al., 1982).
Loss of body weight includes losses in body tissues other than fat.
Therefore, body tissues other than adipose may play a role in
controlling interval to estrus.

In this experiment, lactational ME intake was the most accurate

predictor of interval to estrus. Lactational ME intake has been

71

related inversely to length of the interval from weaning to estrus
(Hughes and Calder, 1979; Reese et al., 1982; King and Dunkin, 1986a).
It is clear that daily ME intake during lactation accounted for the
greatest portion of the variation in postweaning interval to estrus.
It is not clear how the effects of dietary ME influence interval from
weaning to estrus. Body fat, irrespective of dietary ME intake, has a
small effect on the postweaning interval to estrus which indicates
that body fat of primiparous sows is a minor controller of the
interval from weaning to estrus. However, loss of body weight during
lactation accounted for a greater proportion of the variation in
interval from weaning to estrus than body fat.

Thus, effects of dietary ME on postweaning interval to estrus may
be mediated through changes in body composition other than body fat.
Dietary ME appears to control changes in body weight and composition
(O’Grady et al., 1975; Reese et al., 1984). King (1987) postulated
that absolute body protein mass at weaning or relative body protein
loss during lactation determines the postweaning interval to estrus.
Blood urea nitrogen (BUN) has been used as a measure of protein
catabolism. Under equalized intake of dietary protein, BUN has been
used to assess magnitude of muscle catabolism among sows (Reese et
al., 1982; Nelssen et al., 1985). Sows with restricted dietary energy
had delayed postweaning estrus and elevated BUN concentrations
throughout lactation indicating greater muscle catabolism compared
with adequately—fed sows. Sows fed high energy, low protein diets
during lactation lost similar total body weight but less backfat than
sows fed low energy, high protein diets (King and Williams, 1984b;

 

72

Brendemuhl et al., 1987). Thus, composition of weight loss may vary
depending on which dietary essential, energy or protein, is deficient
and which tissue, adipose or muscle is mobilized. Consequently
changes in body composition other than body fat do occur and may
influence the postweaning interval to estrus.

An alternate hypothesis is that dietary ME exerts a direct effect
on postweaning interval to estrus independent of body composition.
There is a negative relationship between lactational energy intake and
length of the anovulatory interval (Figure 8; Reese et al., 1982; King
and Williams, 1984a; King and Dunkin, 1986a). Restricting ME intake
below 10 Mcal/d during lactation resulted in delayed return to
postweaning estrus (King and Dunkin, 1986b). Restricted feeding
results in a myriad of changes in body metabolism. In the adequately-
fed animal, energy is derived from a balanced mixture of dietary
nutrients and body tissues. However, in the restricted-fed animal,
the balance shifts away from dietary nutrients and toward body tissues
as the primary source of energy. This shift in metabolism causes
alterations in circulating concentrations of many metabolites and
hormones such as glucose, ketones, non—esterified fatty acids, BUN,
insulin and growth hormone in humans (Cahill et al., 1966).

Concurrent with these changes in body metabolism, there is a decline
in gonadotropin secretion in rats (Campbell et al., 1977), cattle
(Rutter and Randel, 1984) and swine (Kirkwood et al., 1987) and
reduction of normal activity in the HPO axis which results in
prolonged interval to estrus or anestrus in sows (Armstrong et al.,
1987). One may hypothesize that some hormone, metabolite or catabolic

end product related to the change in body metabolism may affect the

 

 

 

73
interval to estrus. Under this scenario, body composition would have

no direct effect on interval to estrus.

Conclusions
Timing of postweaning estrus in primiparous sows is not dependent
on a minimal threshold of body fat. Furthermore, effects of
lactational ME intake on the interval to estrus are more pronounced
than the effects of body fat. The mechanism by which ME intake

influences postweaning interval to estrus remains to be elucidated.

 

 

 

 

 

Experiment 11
Effect of Stress After Lactation on Postweaning Interval to Estrus

of Primiparous Sows

74

 

 

 

 

 

Introduction

In experiment I, restricting daily ME intake to 7 Mcal during a
28 d lactation caused 87% of sows to display a postweaning interval to
estrus greater than 20 d (Figure 8). In a preliminary experiment
investigating effects of exogenous insulin on postweaning interval to
estrus, sows received a lactational diet identical to the VL diet (7
Mcal ME/d) used in experiment I to create extended intervals to
postweaning estrus in all females. After lactation, sows were
injected daily from weaning until estrus with either insulin or
buffered water (diluent) used to solubilize insulin. All sows were
restrained daily during the treatment period using a hog snare to
obtain blood samples via jugular puncture. The hypothesis was that
exogenous insulin would ameliorate detrimental effects of low
lactational energy intake on interval to postweaning estrus. But, a
large percentage of sows (64%), regardless of postweaning treatment,
displayed a short interval (< 10 d) to estrus indicating either
lactational energy intake was not low enough to create extended
intervals to estrus or some factor imposed in this preliminary
experiment which was not present in experiment 1 induced a prompt
return to estrus. Lactational loss of body weight and backfat of
sows, and litter size and weight at weaning in the preliminary
experiment were similar in magnitude to these same traits in
experiment 1. Genetic background of sows was similar in both

experiments.

 

 

 

76

Therefore, some factor specific to the preliminary experiment may
have caused short intervals to estrus. Two factors were identified as
being present only in the preliminary experiment. First, sows
received daily injections of diluent or diluent containing insulin.
There was no evidence from previous investigations that the diluent
would induce expression of estrus. The second factor specific to the
preliminary experiment was that sows were snared daily for sampling of
blood. This method of handling for sampling of blood represents a
transient, acute stress to pigs (Hemsworth et al., 1981).

Withdrawal of feed and water for 24 h postweaning shortened
interval from weaning to estrus in sows (MacLean, 1969). Mixing gilts
from different social groups and transporting to a new location
increased the proportion of females that display estrus within 4 to 5

d of arrival (Signoret, 1972; Zimmerman, 1976). Braden and Moule

 

(1964) reported transporting ewes via truck or train induced
ovulation. The effects of fasting and transportation on estrus
expression have been attributed to stress resulting from these
activities (Signoret, 1972).

The stress to sows associated with daily snaring and sampling of
blood may have caused an early return to estrus in sows that consumed
7 Mcal ME daily during lactation. The objective of the present
experiment was to determine effects of restraint on interval to

postweaning estrus in primiparous sows.

Materials and Methods
Animals and Management. Fourteen primiparous sows were moved to

farrowing crates in an environmentally-controlled room about 7 d

 

 

77

before parturition. Before parturition, all sows received 1.82 kg
daily of a corn-soybean meal gestation diet (Table 1). Beginning at
parturition, all sows were offered 2.73 kg of'a corn-soybean meal—
solka floc diet (VL diet; Table 1) which provided 7 Mcal ME daily
throughout a 38 i 1.8 d lactation. Calculated intake of all dietary
essentials except ME met or exceeded NRC (1979) recommendations for
lactating sows consuming 4 kg of feed daily (Table 2). This feeding
regimen has been associated with extended intervals to postweaning
estrus in primiparous sows (Experiment I).

Feed intake, body weight and backfat depth of sows were recorded
during lactation as described in experiment I. Litter size for all
sows was adjusted to ten pigs by d 3 postpartum and individual pig
weights were recorded 24 h postpartum and at weaning. Litters did not
receive creep feed.

0n the day of weaning, sows were moved to individual stalls (51
cm x 198 cm) in an environmentally-controlled breeding facility.
Individual sow stalls were connected to an open pen measuring 3.3 m x
3.0 m which housed a mature boar. The boar had continuous fence-line
contact with the genital area of all sows. Beginning one day after
weaning, all sows received gestation diet (Table 1) in an amount based
on their metabolic body weight (kg BW'75) as described in experiment
I.

Sows were assigned based on body weight to one of two postweaning
treatments. Sows assigned to the non-stressed (control) treatment
were backed into the connected boar pen and the boar was permitted to

court sows for 3 to 5 min. After estrus detection was completed, sows

 

 

78

were weighed and returned to their stalls. Sows assigned to the
stressed treatment were handled similar to control sows except that
they were restrained using a hog snare3 and 10 ml of blood was
obtained via jugular puncture. Duration of restraint and sampling of
blood was about one minute. Treatments were imposed daily beginning
one day after weaning until d 35 postweaning. Estrus was recorded
when sows stood to be mounted by a boar and days from weaning to first
estrus were recorded.

Statistical Analysis. Measures of lactational performance of
sows assigned to control and stressed treatments were compared using
least-square analysis of covariance (Gill, 1978) using the procedure
of General Linear Models (SAS, 1982). Body weight and backfat depth
of sows 24 h postpartum were used as covariates for analysis of weight
and backfat depth of sows at weaning, respectively. Covariates
accounted for a meaningful portion (P<.001) of the variation in
response variables. Intervals from weaning to estrus were categorized
into three groups: 510 d in length, > 10 d in length or anestrus to d
35 postweaning. Categories for interval to estrus were cross-
classified with treatments and minimum chi-square was used to

determine degree of independence (Quade and Salama, 1975).

Results
Metabolizable energy intake, body weight and backfat depth of
sows during lactation were not different between control and stressed

sows (Table 6). Similarly, there were no significant differences in

 

3Ring-o-matic hog catcher; Nasco, Fort Atkinson, WI.

 

 

 

TABLE 6. LACTATIONAL PERFORMANCE OF SONS ASSIGNED TO CONTROL OR
STRESSED TREATMENTS DURING THE POSTNEANING PERIOD

79

 

Item

 

 

Control Stressed SEM“

No. of observations 7 7

Lactational metabolizable

energy intake, Mcal ME/d 6.93 i .09 7.08 + 01

Body wt of sows, kg:

24 h pogtpartum 159.0 161.0 7.16

Weaning 124.3 122.3 1.20

Lactational loss 34.7 38.7 1.20
Backfat depth of sows, mm:

24 h postpartum 19.1 19.1 1.75
Weaning 8.7 8.9 .34

Lactational loss 10.4 10.2 34
Litter size at weaning 10.0 9.7 .20
Litter wt at weaning, kg 68.6 69.4 4.29
Weaning to estrus

interval, d 14.2 23.0 5.00

 

“Pooled standard error of the mean.
Length of lactation was 38 i 1.8 d.

 

gig——

 

80

size or weight of litters that suckled control or stressed sows. Sows
that were snared daily to sample blood displayed intervals to first
postweaning estrus similar to non-snared sows (Table 6). Frequency
distribution of weaning to estrus intervals (Table 7) demonstrated a
similar percentage of sows displayed an extended interval to estrus or
were anestrus in control and stressed treatments (4/7 vs 6/7,

respectively).

Discussion

Lack of significant differences in lactational performance of
sows was expected because all sows were fed and managed similarly
throughout lactation. Therefore, a group of sows that were
homogeneous with regard to body weight and backfat depth, previous
nutritional history and intensity of milk production were available
for assignment to postweaning treatments. Body weight and backfat
depth of sows at weaning in this experiment were similar to sows fed
VL diet in experiment I (Table 3). Litter weight on d 21 of lactation
averaged 49.2 kg which is similar to 48.9 kg reported for litters
nursing sows fed VL diet in experiment I.

Fasting sows for 24 h immediately postweaning can shorten
postweaning interval to estrus (MacLean, 1969). The management
practice of mixing gilts from different social groups, transporting,
and relocating to a new pen increases synchrony of estrus expression
(Signoret, 1972; Zimmerman, 1976). Similar effects of transport on
ovulation in ewes has been reported (Braden and Moule, 1964). It has
been generally accepted that stress associated with these management

practices causes the prompt expression of estrus. But, this

 

 

81

TABLE 7. FREQUENCY DISTRIBUTION OF NEANING TO ESTRUS INTERVAL AND
OCCURRENCE OF ANESTRUS FOR PRIMIPAROUS SONS SUBJECTED TO DAILY STRESS

 

Handling treatment

 

 

 

 

Category Control Stressed Total
5 10 days postweaning 3a 1 4
> 10 days postweaning 1 3 4
Anestrusb 3 3 6
Total 7 7 14

 

gNumber of sows. .
Estrus detection lasted 35 d postweaning.

 

 

 

82
mechanism has not been elucidated.

In the present experiment, daily restraint of sows by snaring did
not hasten or delay return to estrus compared with sows that were not
snared. One may conclude that stress imposed by daily snaring of sows
had no effect on interval to estrus. Stress associated with fasting
for 24 to 48 h (King, 1974; Allrich et al., 1979; Tribble and Orr,
1982) or mixing unacquainted sows and moving them to a new pen (Fahmy
and Dufour, 1976) had no effect on interval to estrus. Stress
associated with surgery or illness delayed onset of estrus in gilts
(Hennessy and Williamson, 1983). Delay in onset of estrus can be
reproduced by administration of corticosteroids (Liptrap, 1970) or
adrenocorticotrophin (Liptrap, 1970; Hennessy and Williamson, 1983) to
non-stressed gilts indicating that delayed estrus may be due to
elevated concentrations of stress responsive hormones. In contrast,
unpleasant handling of gilts increased concentrations of
corticosteroids in plasma but had no effect on expression of estrus
(Hemsworth et al., 1986).

The inconsistent effects of stress on expression of estrus may be
explained by our limited understanding of effects of stress on
livestock. There has been no correlation between hormonal or
behavioral changes and adverse effects on animal productivity (Moberg,
1987). Another explanation for inconsistent effects of stress is that
stress is not generic. The stress associated with fasting may be
different from that associated with transportation or physical
restraint. From this, one may hypothesize that response to different

stressors may be heterogeneous (Gibbs, 1985). One cannot be certain

 

 

83

that treatments designed to stress sows achieved their goal. In the
present study, corticosteroid concentrations in plasma were not
quantified to determine if physical restraint by snaring elicited a
stress response in sows. But, the stressed treatment mimicked
conditions of the preliminary experiment in which snared sows

displayed an unexpected short interval to estrus.

Conclusions
Stress induced by daily snaring and sampling of blood during the
postweaning period did not influence interval to estrus. Thus, the
uncharacteristic short intervals to estrus of sows in the preliminary
insulin experiment were probably not caused by the daily routine of

collecting blood.

 

 

 

 

Experiment 111

Effects of Exogenous Insulin After Lactation on Postweaning Interval

 

to Estrus of Primiparous Sows

 

84

85

Introduction

Severe feed restriction causes cessation of normal estrous cycles
in female livestock (Imakawa et al., 1986a; Armstrong and Britt,
1987). Restricted feeding elicits alterations in concentration of
blood borne metabolites (Cahill et al., 1966; Wangsness et al., 1981;
Reese et al., 1982) which presumably reflect adjustments in body
metabolism. However, the relationship between nutritionally-induced
changes in body metabolism and expression of estrus has not been
elucidated.

Insulin is a hormone of central importance to homeostatic
regulation of metabolism in mammalian organisms. Restricted
feeding depresses concentration of insulin in blood (Kasuga et al.,
1977; Wangsness et al., 1981; Armstrong and Britt, 1987) while
realimentation increases concentration of insulin in blood to levels
indicative of the "fed" state (Cahill, 1971; Armstrong and Britt,
1987). Low concentration of insulin in blood associated with
restricted feeding may be responsible for cessation of estrous cycles
in restricted-fed females. Depressed concentration of insulin in
blood of insulin-dependent diabetics is associated with primary
amenorrhea and anovulation in women (Poretsky and Kalin, 1987) and
prolonged estrous cycles, anestrus and anovulation in rats (Liu et

.al., 1972; Kirchick et al., 1978). Exogenous insulin restores normal

reproductive functions in diabetic females (Poretsky and Kalin, 1987).

Insulin positively affects endocrine glands of the HPO axis.

 

 

 

86

Insulin increased basal and GnRH-stimulated release of LH and FSH from
cultured anterior pituitary cells (Adashi et al., 1981). Furthermore,
insulin is necessary for development of optimal steroidogenic
potential of granulosal cells (May et al., 1980; Lino et al., 1985).
Exogenous insulin increased ovulation rate of gilts (Cox et al., 1987)
and heifers that were in negative energy balance (Harrison and Randel,
1986) indicating that insulin has gonadotropic effects. The mechanism
responsible for the insulin-induced increases in ovulation rate has
not been determined. One possibility is that insulin increases
secretion of gonadotropic hormones which stimulate follicular growth.
Several workers have reported that exogenous insulin increases mean
concentration of LH in serum of beef cows (Earnest et al., 1988) and
pulsatile release of LH in gilts (Cox et al., 1987; Britt et al.,
1988). However, the LH response to exogenous insulin is inconsistent
(Cox et al., 1987). Frequency of LH pulses increases after weaning in
sows (Shaw and Foxcroft, 1985; Foxcroft et al., 1987). Increased
frequency of LH pulses seem to be the signal which stimulates estrus
expression after an extended anestrous period (Haresign et al., 1983).

One may hypothesize that low concentrations of insulin in blood
associated with restricted feeding do not stimulate the HPO axis
adequately to elicit resumption of ovarian cyclicity after lactation
in sows. Therefore, the main objective of this experiment was to
determine if exogenous insulin would hasten return to postweaning
estrus in restricted-fed sows. A secondary objective was to examine

the relationship of selected blood-borne metabolites to occurrence of

postweaning estrus.

 

 

87
Materials and Methods

Animals and Management. About 7 d before parturition, twenty six
primiparous sows were moved to farrowing crates in an environmentally
controlled room. Before parturition, all sows received 1.82 kg daily
of a corn-soybean meal based gestation diet (Table 1). Beginning at
parturition, all sows received 2.73 kg of a corn—soybean meal—solka
floc diet (VL diet; Table 1) which provided 7 Mcal ME daily throughout
lactation. Mean length of lactation was 34.8 i 1.3 d. Calculated
daily intake of all dietary essentials except ME met or exceeded NRC
(1979) recommendations for lactating sows consuming 4 kg of feed daily
(Table 2). This feeding regimen provided 58% of recommended daily ME
intake and has been associated with extended intervals to postweaning
estrus in primiparous sows (Experiment I).

Feed intake, body weight and backfat depth of sows were recorded
during lactation as described in experiment I. Litter size was
adjusted to ten pigs by d 3 postpartum and individual pig weights were
recorded 24 h postpartum and at weaning. Pigs did not receive creep
feed and access to the sow’s feed was minimal.

On the day of weaning, sows were moved to individual stalls in an
environmentally—controlled breeding facility and assigned to one of
two postweaning treatments. Beginning with the first sow to be
separated from her litter, every second sow was assigned to the
control group (n=13) while remaining sows were allotted to the

experimental group (n=13). This schedule ensured that control and

 

experimental sows were distributed equally throughout the entire

experiment which began on March 5, 1988 and concluded on

 

 

September 20, 1988.

Sows assigned to the experimental group received a daily

intramuscular injection of .75 IU insulin4

per kg body weight at about
0900 h. Control sows were not injected. Treatment with insulin began
the day after weaning and continued until estrus or d 30 postweaning,

whichever occurred earlier.

Beginning the day after weaning, all sows received gestation
diet (Table 1) in an amount based on their metabolic body weight (kg
BW°75) as described in experiment I. Sows were weighed daily to
determine their daily allotment of feed. Feed was offered once daily
at 1600 h. Estrus detection using a mature boar was performed daily
at 0900 h from the day after weaning until d 30 postweaning. Sows
were considered to be in estrus when they stood for mating. Days
from weaning to first estrus were recorded.

Blood was sampled twice weekly via jugular puncture beginning the
day of weaning and continuing until estrus was detected or d 30
postweaning whichever occurred earlier. Collection of blood during
the postweaning period occurred at 0900 h immediately before injection
of insulin. Therefore, blood was sampled 24 h after the previous
injection of insulin and about 18 h postprandial. Once during the
postweaning period, blood was collected from five control and six
insulin treated sows at 2 h intervals for 24 h to characterize insulin
and glucose concentrations. Sows selected for characterization of
diurnal insulin and glucose concentrations had either expressed estrus

——:—-—=—__

4Protamine, zinc and Iletin 1, derived from beef and pork pancreas.
Eli Lilly and Company, Indianapolis.

 

 

 

 

 

 

89

(one control and one insulin-treated sow) or had been separated from
their litters at least 15 d (range 16 to 41 d) and had not expressed
estrus. Sows were maintained on their respective treatment regimens
until intensive blood sampling was completed. At each sampling, three
milliliters of blood were collected in evacuated tubes containing
anticoagulant (potassium oxalate) and glycolytic inhibitor (sodium
fluoride) and plasma was separated by centrifugation within 3 h of
collection. An additional ten milliliters of blood were allowed to
clot for 24 h at 4°C; then serum was harvested by centrifugation.
Serum and plasma were stored at -20°C until hormone or metabolite
assays were performed.

Hormone and Metabolite Assays. Porcine insulin in serum was
quantified using a heterologous double antibody radioimmunoassay with
methods similar to Villa-Godoy (1987). In this assay, antisera (first
antibody) to bovine insulin5 (lot GP23) were produced in guinea pigs
and anti—guinea pig gamma globulin (second antibody) was produced in
sheep (Tucker, 1971). Radioiodination of bovine insulin6 was as
described by Villa-Godoy (1987). Dilution of antisera to l:30,000 in
guinea pig control sera7 (1:400 in .05 M EDTA—phosphate buffered
saline; pH 7.0) at a volume of 200 ul per tube provided approximately
25% specific binding of 125I-insulin and was used in assay and
validation procedures. Specificity of the antisera to bovine insulin
was determined by testing degree of cross-reactivity with bovine

growth hormone (purified, 15825-AJP-152)8, bovine luteinizing hormone

 

zMiles Scientific, Naperville, IL.

7Novo Biolabs, Wilton, CT.
8Gibco, Grand Island, NY.
The Upjohn Company, Kalamazoo, MI.

 

 

 

 

 

 

90
(NIH 88), bovine follicle stimulating hormone (USDA 8P3), bovine

thyroid stimulating hormone (NIH BS), bovine prolactin (NIH B4) or
bovine-porcine glucagon9 in amounts up to 50 ng/tube. Cross-
reactivity was less than 1% for each hormone. Standard curves
contained .025, .05, .10, .15, .20, .30, .40, .50, .60, 1.00, 2.00,
3.00 and 4.00 ng porcine insulin per tube. Standard curves,
calculated from multiple regression equations with linear, quadratic
and cubic components, had coefficients of determination ranging from
.990 to .997. Porcine serum was supplemented with 2.5 and 5.0 ng
porcine insulin9 per tube (10 replications each) and assayed according
to methods of Villa-Godoy (1987) to determine accuracy. After
correction for endogenous insulin in serum, content of insulin
measured in each tube did not differ from exogenous insulin. The
dilution response curve (40, 60, 80 and 100 ul/tube) was parallel to
the porcine insulin standard curve. All samples were quantified in a
single assay. The intra-assay coefficient of variation was 7.0%

for a serum sample containing .323i.007 ng insulin/ml and 17% for a
serum sample containing 1.8151.319 ng insulin/ml. Lower limit of
sensitivity was .09 ng porcine insulin/ml.

Exogenous bovine and porcine insulin stimulates production of
endogenous antibodies against insulin in humans (Berhanu and Olefsky,
1985). These endogenous antibodies may interfere with binding of
first antibody to insulin in the radioimmunoassay. To determine if

insulin-treated sows produced antibodies against exogenous insulin,

 

9Sigma Chemical Company, St. Louis, MO.

 

 

 

 

 

 

 

 

91
200 ul of serum from each insulin-treated sow was used in place of first

antibody. Second antibody was anti-porcine gamma globulin10 (200 ul;
diluted 1:5) produced in rabbits. Binding of radioiodinated insulin
was less than 5% indicating serum from insulin-treated sows contained
no endogenously produced antibodies to exogenous insulin.

Progesterone concentrations were determined in one plasma sample
per week per sow by radioimmunoassay (Experiment I). Intra-assay and
inter-assay coefficient of variation was 7% and 3%, respectively for
standard sera containing .31 i .04 ng progesterone/ml.‘ Presence of
functional corpora lutea was assumed when concentration of
progesterone in plasma was >1 ng/ml. Blood was sampled only from sows
not detected in estrus, therefore elevated plasma progesterone
indicated ovulation in the absence of behavioral estrus.

Enzymatic colorimetric procedures were used to quantify

11, urea nitrogen12 (UN) and non—esterified

concentrations of glucose
fatty acids13 (NEFA) in plasma.

Statistical Analyses. Measures of lactational performance of
sows assigned to control and insulin treatments were compared using
the General Linear Models procedure (SAS, 1982) for least-squares
analysis of covariance (Gill, 1978a). Body weight and backfat depth
of sows 24 h postpartum were used as covariates for analysis of weight

and backfat depth of sows at weaning, respectively. Covariates

accounted for a meaningful portion (P<.001) of the variation in

 

10

11ICN Immunobiologicals, Lisle, IL.

Glucose (Trinder), procedure no. 315, Sigma Chemical Company., St.

Louis.

12Urea nitrogen, procedure no. 535, Sigma Chemical Company, St.
ouis.

1 NEFA C, Wako Pure Chemical Industries, Ltd., Osaka, Japan.

 

 

 

 

 

92
response variables. Intervals from weaning t0 estrus were categorized

into three groups as described in experiment 11. Categories of
interval to estrus were cross-classified with insulin treatments and
minimum chi-square was used to determine degree of independence (Quade
and Salama, 1975).

The experimental period began the day after weaning and ended
when sows displayed estrus or 30 d postweaning whichever occurred
earlier. Consequently, the duration of treatment for each sow
represented 2 to 10 blood samples under a twice weekly schedule of
sampling. A regression line containing linear, quadratic and cubic
components was fitted to data on insulin concentration in blood for
each sow to address the unbalanced nature of these data. To determine
effects of exogenous insulin on concentration of insulin in serum,
multivariate analysis of variance was performed on components of the
polynomial regressions (SAS, 1982). Five sows (2 control and 3
insulin-treated) expressed estrus by d 7 postweaning which allowed
collection of 5 3 blood samples from these sows. The small number of
samples did not permit calculation of a cubic polynomial for these
sows. Therefore, data from 21 sows were used in multivariate analysis
of variance procedures. This multivariate procedure was repeated to
determine effects of exogenous insulin on concentration of glucose, UN
and NEFA in plasma. Concentrations of insulin and glucose in blood
samples collected every 2 h for 24 h were analyzed as a split-plot
experiment with repeated measurements in time (Gill, 1978b).

To further understand the relationship among blood-borne insulin

and metabolites and expression of estrus, sows were divided into four

 

 

 

 

93
groups: 1. control sows that displayed estrus by d 30 postweaning

(control—cyclic), 2. control sows that were anestrous through d 30
postweaning (control-acyclic), 3. insulin-treated sows that displayed
estrus (insulin-cyclic), and 4. insulin-treated sows that were
anestrous (insulin—acyclic). Concentrations of insulin, glucose, UN
and NEFA in blood collected at weaning and at the last sampling time
(53 d before estrus or d 30 postweaning) were compared using least-
squares analysis of variance. The statistical model for this analysis
contained the main effects of insulin treatment (control or insulin),
ovarian status (cyclic or acyclic) and the interaction of main
effects. In all statistical analyses, sow was considered the

experimental unit. All reported means are least squares means.

Results

Metabolizable energy intake during lactation, body weight and
backfat depth 24 h postpartum and at weaning, litter size and litter
weight at weaning were not different between control and insulin-
treated sows (Table 8). Concentrations of insulin in serum of one
insulin-treated sow, that was determined to be an outlier (P<.01) by
the procedures of Gill (1978a), were removed from the mean profile of
insulin concentrations during 24 h post-injection. Sows injected once
daily with .75 IU insulin per kg body weight had greater (P<.05)
concentrations of insulin in serum throughout the ensuing 24 h
compared with non-injected sows (Figure 9). Concentration of insulin
in serum rose steadily after injection until it reached a peak at 1800
h. Elevated concentration of insulin depressed (P<.05) concentrations

of glucose during 24 h post-injection in plasma of insulin—treated

 

 

 

i I‘d}, T '-

 

 

 

TABLE 8. LACTATIONAL PERFORMANCE OF SONS ASSIGNED TO CONTROL OR

INSULIN TREATMENTS

 

 

Insulin

Item Control treated SEMa
N0. of observations 13 13

Lactational metabolizable

energy intake, Mcal ME/d 7.01 7.04 .04
Body wt of sows, kg:

24 h postpartum 167.1 173.4 4.27
Weaning 133.5 133.2 1.57
Backfat depth of sows, mm:

24 h postpartum 19.6 21.5 .80
Weaning 10.2 10.4 .47
Litter size at weaning 9.7 9.4 .21
Litter wt at weaning, kg 69.4 69.0 2.64

 

“Pooled standard error of the mean.
Length of lactation was 34.8 i 1.3 d.

 

 

 

 

 

Figure 9.

 

Serum insulin log/ml)

 

 

2.0

1.5

1.0

 

951

J L I I I l L J #1 I

 

 

 

 

(10
BO Plasma glucose img/dll

70 " *
I *§ §

60- A

I 50

40*-
Trt (P < .05l

 

30)-
l

t L _I_ __I_ ’I I I J__ L I 21*

 

 

 

20 3*
1000 1400 1800 2200 200 600
Time of day

--- Control -+- insulin treated

Concentration of insulin in serum (panel A) and glucose

in plasma (panel B) of control and insulin-treated sows during
24 h after injection of insulin. Vertical arrows in panel B
designate time of injection (1005 h) and feeding (1605 h).
Asterisks designate significant differences between treatments

within time of sampling (*P<.O5; **P<.01).

 

 

 

 

96

sows compared with control sows. Following injection of insulin at
1005 h, plasma glucose declined to nadir at 1600 h. Sows were offered
their daily meal at 1605 h which elicited a postprandial rise in
glucose concentration observed at 1800 h. By 2000 h, plasma glucose
concentration of insulin—treated sows was lower (P<.05) than control
sows and slowly increased throughout the remainder of the sampling
period.

Insulin-treated sows had higher (P<.05) concentrations of insulin.
in serum collected 24 h after injection throughout the postweaning
period compared with control sows (Figure 10). Elevated serum insulin
was associated with lower (P<.02) concentration of glucose in plasma
collected 24 h post-injection from insulin-treated sows compared with
controls. Based on data presented in Figures 9 and 10, intramuscular
injection of insulin chronically elevated serum insulin and depressed
plasma glucose concentrations. Therefore, an evaluation of effects of
exogenous insulin on postweaning expression of estrus was possible.
Concentration of NEFA or UN in plasma collected 24 h after injection
of insulin was not different between insulin-treated and control sows
(data not shown).

Exogenous insulin delayed (P<.05) postweaning estrus in
primiparous sows (Minimum chi—square = 7.23; Table 9). A greater
proportion of insulin-treated sows were anestrous 30 d postweaning
compared with control sows. Concentration of progesterone in plasma
samples collected weekly from sows that failed to display estrus by d
30 postweaning did not exceed 1 ng/ml. Therefore, corpora lutea were

not present in anestrous sows indicating that these females were

 

 

 

 

97

Serum insulin (rig/mil

 

 

 

 

 

 

 

 

 

 

A\

1.5 - _
1.0 *-
05 - Ti‘t (P < .05) ’
0.0 l J I L I L _.I I

Plasma glucose img/dil
80
70 F B

1 l
50i'
50 .—
40 .-
30 *- Trt (:3 < .02)
20 4 1 i— 1 1 L I L
Weaning 3 7 10 i4 17 21 24 28 31

Time after weaning (d)

-*- Control —+— insulin treated

Figure 10. Concentration of insulin in serum (panel A) and glucose
in plasma (panel B) collected 24 h postinjection during the
postweaning period from control and insulin-treated sows.

98

TABLE 9. EFFECT OF EXOGENOUS INSULIN 0N FREQUENCY DISTRIBUTION OF
NEANING T0 ESTRUS INTERVAL AND OCCURRENCE OF ANESTRUS FOR PRIMIPAROUS

 

 

 

SOWS
Category Control Insulin treated Total
5 10 d postweaning 2a 3 5
> 10 d postweaning 6 0 6
Anestrusb 5 10 15
Total 13 13 26

 

 

gNumber 0f sows.
Estrus detection lasted 30 d postweaning.

 

99

anovulatory.

Sows were divided by insulin treatment and ovarian status into
four groups to investigate the relationship between body metabolism
and expression of estrus. Mean interval to estrus for all cyclic sows
was 13.9:8.9 d. Graphic characterization of insulin and metabolite
concentration in blood of sows during the postweaning period is
presented in Figures 11 to 14. Endpoint of this experiment was day of
estrus expression or d 30 postweaning whichever occurred earlier;
consequently, the number of observations at each day after weaning in
panel A of Figures 11 to 14 progressively declined as sows expressed
estrus and were removed from the experiment.

There were no interactions between insulin treatments and ovarian
status for concentration of insulin or metabolites in blood, so only
main effects will be discussed. Concentrations of insulin, glucose
(Figures 11 and 12) or UN (Figures 13 and 14) in blood on the day of
weaning or at the end of the experiment was not affected by insulin
treatment or ovarian status (Table 10). However, the concentration of
NEFA on the day of weaning (P<.06) and at the end of the experiment
(P<.01) was greater in cyclic sows than in acyclic sows (Table 10).
Insulin treatment had no effect on concentration of NEFA at weaning or
at termination of the experiment.

Mean concentration of insulin in serum during the postweaning
period was not different between insulin—treated and control sows
(Table 11). Mean concentration of glucose during the postweaning
period tended (P<.09) to be lower in insulin-treated sows than in
control sows (Table 11). Ovarian status had no effect on mean

concentration of insulin or glucose during the postweaning period.

 

 

 

100

 

 

 

 

 

 

 

 

Serum insulin log/mil Plasma Glucose img/dli
2'0 , 70
W A ~ 60
1.5
n=8 7 6 6 e 5 3 2 - 50
10- ~40
~ 30
0.5 i‘
‘20
91—0" 1 *1 I I I I I IL 10
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AW
"60
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“'50
LOF- ~40
i J 30
0.5 _ D
"20
0.0 I L I_ _L J Jfi _I l 10
Weaning a 7 1o 14 17 21 24 28 31

 

Time after weaning id)

-°- insulin -+- Glucose

 

Figure 11. Concentration of insulin and glucose in blood of control-
cyclic (panel A) and control-acyclic (panel B) sows during the
postweaning period.

 

 

101

 

 

 

 

 

 

 

 

 

 

 

Serum insulin hg/mll Plasma Glucose img/dli
2!) 70
n<3 £3 1
i\ A - so
1£5r -\\\\
~50
1-0 ' - 4o
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50
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"20
0.0 J 1 A“ L L ._1w_ 1— _! 1°
Naming 3 7 1o 14 17 21 24 as 31

Time after weaning (d)

'**-lnsuun '“F-Eflucose

Figure 12. Concentration of insulin and glucose in blood of insulin-
cyclic (panel A) and insulin-acyclic (panel B) sows during the
postweaning period.

102

 

 

 

 

 

 

400 NifA “139/" Urea nitmgen img/dll

7 a 5 a 5 3 2 5°
350

/\
300 " _ 40
250-
200- \t - so
150-
100 ‘ 1 ' I l L 1 I 20
400 50
350* [1.5
B
300 — _ 40
250- /Ax\ «“/T‘\~
, \‘ __ ‘7‘
P§\‘ _,,4(’ ”+—” ~4(//
200 - \t—fii/ - 30
150-
100 I I l L I I I l
Waning 3 7 1o 14 17 21 24 as 31
Time after weaning id)
H~ NEFA ~+~ Urea nitrogen
Figure 13. Concentration of non—esterified fatty acids (NEFA) and

urea nitrogen in plasma of control—cyclic

(panel A) and control-

acyclic (panel B) sows during the postweaning period.

 

 

103

NEFA iueq/ll Urea nitrogen [mg/d!) 50

 

400
ms 3 1
A

 

 

 

 

§
88

350’ n-iO , B

 

 

 

 

 

 

Waning 3 7 10 14 17 21
Time after weaning (d)

H- NEFA -+- Urea nitrogen

Figure 14. Concentration of non—esterified fatty acids (NEFA) and
urea nitrogen in plasma of insulin-cyclic (panel A) and insulin-
acyclic (panel B) sows during the postweaning period.

 

104

 

 

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105

 

 

 

Afio.vav
msumpm :m_cw>o

 

 

 

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oonma wsz<m3Pmoa mIH wszzo GOOAm zH mmHHgom<pmz ombumgmm oz< ZHADWZH no onF<mHzmozoo z<mz .HH m4m<H

 

 

106
Concentration of UN in plasma tended to be lower in insulin-treated
sows (P<.06) and sows that expressed estrus (P<.09) compared with
control and anestrous sows, respectively (Table 11). Mean
concentration of NEFA during the postweaning period was increased by
exogenous insulin (P<.05). Similarly, sows that displayed estrus had
greater (P<.01) mean concentration of NEFA in plasma throughout the
postweaning period than anestrous sows.

Energy intake of sows during lactation was not affected by
insulin treatment or ovarian status (Table 12). Cyclic sows were
heavier (P<.05) and had more backfat (P<.05) 24 h postpartum and at
weaning compared with acyclic sows. Differences in body weight and
backfat depth at weaning were not apparent when adjusted for
differences in body weight and backfat depth 24 h postpartum by
covariate analysis of variance. Loss of weight and backfat depth
during lactation were similar among the four groups of sows.
Likewise, litter weight at weaning was not influenced by insulin
treatment or ovarian status.

Discussion

Measures of sow performance during lactation were not different
between control and insulin-treated sows. This was expected because
all sows were fed to managed alike during lactation. Similarity of
performance during lactation indicates that nutritional management
during lactation yielded a homogeneous group of sows that could be
assigned to postweaning treatment.

Sows injected with .75 IU protamine zinc insulin per kg body

weight once daily experienced greater concentration of insulin in

M- T

 

 

 

 

107

 

 

 

 

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cmcz pcmcmuwwu pa: mcmmzn
.coccm ugmucmpm H.cmwzm

 

 

 

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umpmwcp cv_=mcH _ocp:ou

 

m2H<Hm z<Hm<>o oz< hzmzh<mmk zHAszH OH wz~omouo< omHmHmm<AQ onH<Hu<4 wszDQ mzom mo muz<zmommma .NH m4m<k

 

 

 

108

serum during the 24 h post-injection compared with control sows
(Figure 9). The small peptide, protamine, complexed to insulin causes
a slow, sustained release of insulin from the site of injection (Dr.
G. Sampson, Eli Lilly Co., personal communication). This slow release
maintained elevated serum insulin concentrations over an extended
period of time.

In response to elevated serum insulin, mean concentration of
plasma glucose during the 24 h post-injection was depressed in
insulin-treated sows compared with control sows. Plasma glucose
declined steadily from injection of insulin until feeding at 1605 h.
Feeding increased glucose 2 h postprandial but by 4 h after feeding,
plasma glucose of insulin-treated sows returned to a concentration
significantly lower than control sows. Throughout the remainder of
the sampling period, concentration of glucose in insulin—treated sows
slowly increased to a concentration that was not different from
control sows. Slowly increasing plasma glucose concentrations
concurrent with elevated concentration of insulin in serum would
indicate that insulin-treated sows experienced some degree of insulin
resistance during the last 10 h of sampling. Insulin-induced
hypoglycemia can produce insulin resistance in humans for up to 7 h
after euglycemia is achieved (Clore et al., 1987; Kollind et al.,
1988). The data presented in Figure 9 provide evidence that the daily
im injection of protamine zinc insulin increased concentration of
insulin in serum for up to 24 h. Furthermore, the dose of insulin
administered had sufficient biological activity to significantly
depress mean concentration of glucose in plasma during the 24 h after

injection.

 

 

 

 

109
Concentration of insulin in serum collected 24 h post-injection

throughout the postweaning period in insulin-treated sows was greater
than in control sows (Figure 10). Exogenous insulin had a prolonged
effect on body metabolism of insulin-treated sows because plasma
glucose was depressed significantly in blood collected 24 h after
injection compared with that of control sows. From these data, one
can conclude that insulin-treated sows were exposed to chronically
elevated serum insulin concentrations.

Despite chronically elevated concentration of insulin in serum,
exogenous insulin did not shorten the postweaning interval to estrus
(Table 9). A greater percentage of insulin-treated sows failed to
express estrus by d 30 postweaning compared with control sows. One
may question whether the daily dose of exogenous insulin in this
experiment was high enough to elicit a positive effect on postweaning
expression of estrus. In a preliminary trial, 1.0 IU protamine zinc
insulin per kg body weight caused death or clinical signs of severe
hypoglycemia in 100% of treated sows after about 5 d of treatment.
Signs of clinical hypoglycemia such as head pressing, circling,
convulsions and hyperthermia were observed in 38% of sows that
received .75 IU protamine zinc insulin per kg body weight. The
sporadic occurrence of clinical hypoglycemia in the present experiment
would suggest that .75 IU protamine zinc insulin per kg body weight
was the maximal tolerable dose for sows under the conditions of this
experiment.

Many workers have reported that exogenous insulin exerts positive

effects on the HPO axis (see Review of Literature). It is not clear

 

 

 

 

 

110
why administration of insulin in the present experiment did not affect

positively expression of estrus postweaning. Four factors can be
identified that potentially explain lack of a positive effect of
exogenous insulin on postweaning expression of estrus.

Elevated concentration of insulin in blood can reduce the number
of receptors for insulin in various tissues (Olefsky, 1976; Galbraith
et al., 1981). Chronic hyperinsulinemia in rats lowered specific
binding of insulin to ovarian tissue homogenate suggesting receptors
for insulin in ovarian tissue can be down-regulated by
hyperinsulinemia (Poretsky et al., 1988). Similarly, hyperinsulinemia
did not increase binding of insulin to receptors in hypothalami of
rats (Melnyk and Martin, 1984). Perhaps hyperinsulinemia of insulin-
treated sows caused down-regulation of insulin receptors in the HPO
axis such that potentially positive effects of exogenous insulin on
the HPO axis were not expressed.

Slowly increasing concentration of glucose concurrent with
elevated concentration of insulin in serum during the period from 12
to 24 h after injection of insulin suggests that sows became resistant
to insulin as discussed earlier (Figure 9). Insulin resistance would
reduce the biological effect of exogenous insulin. Therefore, even
though serum insulin was elevated, biological effectiveness of
exogenous insulin on postweaning expression of estrus may have been
diminished.

Dietary energy may modulate effects of exogenous insulin on the
HPO axis. Cox et al. (1987) reported that exogenous insulin increased
ovulation rate in gilts fed diets adequate in energy (5770 kcal ME

daily). Response to exogenous insulin was increased when gilts

 

 

 

 

111
received a high energy diet (9960 kcal ME daily). In contrast, gilts
made anestrous by dietary restriction (480 kcal ME daily) responded to
exogenous insulin with increased pulse frequency of LH and increased
number of small follicles (Britt et al., 1988). However, insulin did
not stimulate follicular development enough to cause resumption of
estrous cycles. Response to exogenous insulin may be modulated by
energy intake in the period before or during administration of
insulin. In the present experiment, sows received a fortified diet
during the postweaning period at levels calculated to meet maintenance
requirements for all nutrients and energy. Feed intake of sows during
the postweaning period or severe energy restriction during lactation
may have limited a positive effect of insulin on the HPO axis and
expression of estrus.

A final potential explanation for lack of a positive effect of
insulin on estrus expression is that exogenous insulin may not be
gonadotropic 0r stimulatory to the hypothalamus or anterior pituitary
in sows. Such a conclusion would be premature given that this is the
first report of effects of exogenous insulin on postweaning interval
to estrus in sows.

Concentration of insulin in serum declined after weaning in all
sows except insulin-treated sows that displayed estrus (Figure 11 and
12). Other researchers reported concentrations of insulin in blood of
sows increased after pigs were weaned (Armstrong et al., 1986;
Eriksson et al., 1987). Concentrations of insulin in serum in the
present experiment are confounded with time of sampling. Blood was

sampled 2 to 3 h postprandial on day of weaning while subsequent

 

 

112
samples were collected about 7 h preprandial. Therefore,
concentration of insulin on day of weaning probably represents a meal-
induced surge of insulin while subsequent samples represent baseline
preprandial concentrations. Regardless of confounding effects of time
of sampling, concentration of insulin at weaning was not different
between cyclic and acyclic sows (Table 10). Unfortunately, a similar
comparison of preprandial or basal insulin concentration on day of
weaning cannot be accomplished. Concentrations of insulin at end of
the experiment (estrus or d 30 postweaning) and mean concentrations of
insulin in serum during the postweaning period were not different
between cyclic and acyclic sows (Tables 10,11). These data further
support the conclusion that insulin did not have a stimulatory effect
on HPO axis of sows during the postweaning period.

Insulin-treated sows experienced elevated serum insulin,
depressed plasma glucose and a higher incidence of anestrus compared
with control sows. Exogenous insulin and associated depressed plasma
glucose has been related to lengthened estrous cycles in dairy cows
(McClure, 1968). It has been proposed that plasma glucose may control
return to ovarian cyclicity and expression of estrus (McClure, 1968;
McClure et al., 1978; Short and Adams, 1988). If this is true,
depressed concentration of glucose in plasma of insulin-treated sows
may explain the higher incidence of anestrus caused by exogenous
insulin. An association among restricted feeding, depressed blood
glucose concentration and anestrus has been established in cattle
(McCann and Hansel, 1986). Depressed plasma glucose caused by
elevated renal clearance of glucose negatively affected LH secretion

in beef cows (Rutter and Manns, 1988). However, a direct cause and

 

113
effect relationship between depressed blood glucose and occurrence of
anestrus has not been established. Concentrations of glucose in
plasma of sows at weaning, at the end of the experiment or mean
concentration of plasma glucose during the postweaning period were not
different between cyclic and acyclic sows (Tables 10 and 11).
Therefore, one may conclude that plasma glucose does not account for a
significant portion of the variation in postweaning expression of
estrus.

Urea nitrogen in blood results from catabolism of protein.
Catabolized protein may be derived from diet or body tissues. Under
standardized intake of dietary protein, UN has been used as a crude
measure of muscle catabolism (Reese et al., 1982; Nelssen et al.,
1985). Elevated UN is associated with restricted feeding and delayed
return to postweaning estrus (Reese et al., 1982; Nelssen et al.,
1985). In previous experiments, relationship between UN and
expression of postweaning estrus has been confounded by differences in
feed intake and loss of body weight. In the present experiment, UN
was not different between cyclic and acyclic sows at weaning or at end
of the experiment (Table 10; Figures 13 and 14). Cyclic sows tended
to have lower (P<.09) mean concentrations of UN during the postweaning
period than acyclic sows (Table 11). Similar results have been
reported for restricted-fed sows by others (Reese et al., 1984).

Similar concentration of UN between cyclic and acyclic sows that were
restricted-fed would suggest that the extent of muscle catabolism was
not different. The evidence is weak that UN is a controller of

postweaning expression of estrus. This conclusion is not clouded by

 

 

 

 

 

114
differences in dietary intake of protein because sows were fed
similarly throughout the experiment.

Greater concentrations of NEFA in plasma of sows that displayed
estrus compared with anestrous sows are puzzling. Assuming
concentrations of NEFA in plasma reflect mobilization of adipose
tissue; one would expect elevated NEFA to be associated with increased
loss of body weight or backfat. Concentrations of NEFA in blood
collected at weaning were not associated with loss of body weight
(r2=.20) 0r backfat (r2=.07) during lactation (Table 12).
Concentrations of NEFA in plasma have been associated positively with
yield of milk in dairy cows (Athanasiou and Phillips, 1978). Litter
weight at weaning was not different between cyclic and acyclic sows
indicating that milk yield during lactation was similar between the
two groups of sows.

Other workers have reported that sows which expressed prompt
postweaning estrus had higher NEFA concentration in plasma during mid-

lactation compared with sows that experienced delayed return to estrus

 

(Armstrong et al., 1986). Concentrations of NEFA at weaning were not
different between early- and late-cycling sows. Likewise, weight and
backfat change and litter weight gain during lactation were similar
between groups of sows. Elevated NEFA concentrations in plasma have
been associated with reduced fertility in dairy cows (Ducker et al.,
1985). However, this relationship was confounded by lower energy
balance of cows that experienced reduced fertility. Energy balance as
indicated by change in weight and backfat of sows and litter weight at
weaning was not different between cyclic and acyclic sows in the

present experiment.

 

 

 

 

115

In contrast to loss of body weight and backfat during
lactation, absolute body weight and backfat depth at weaning were
greater in cyclic than in acyclic sows. Body weight alone does not
explain a significant portion of variation in expression of estrus
postweaning (Reese et al., 1984; Johnston et al., 1987). Backfat
depth is correlated positively with body fat (Whittemore et al., 1980;
Knudson et al.,1985) and can be used as an indicator of total body
fat. But, backfat depth (Johnston et al., 1987) and body fat
(Experiment I) at weaning explain a small portion of variation in
postweaning interval to estrus. Armstrong et al. (1987) reported that
estrous cycles resumed upon realimentation of nutritionally anestrous
gilts without a significant increase in backfat. One cannot state
definitively that body fat did not influence expression of estrus, but
it seems unlikely that estrus expression of cyclic sows was due to
larger body fat reserves than that of acyclic sows. Furthermore,
concentrations of NEFA in plasma are not related to body fatness (Wood
et al., 1977; Wangsness et al., 1981; Villa-Godoy, 1987). Therefore,
NEFA in plasma may affect postweaning expression of estrus independent
of body composition.

It is not clear why concentration of NEFA in plasma of cyclic
sows was elevated. Elevated NEFA in plasma can result from increased
mobilization of fatty acids from adipose tissue, greater rate of fat
absorption from the gastrointestinal tract, decreased clearance rate
of NEFA from plasma or a combination of these phenomena. The exact
reason for elevated NEFA cannot be determined in this experiment.

But, absorption of fat from gastrointestinal tract probably does not

 

 

 

 

 

 

116

explain elevated plasma NEFA because all sows received the same diet.
It is clear that elevated NEFA at weaning and during the postweaning
period was associated with expression of estrus. Possibly, NEFA
provide additional energy to tissues of the HPO axis which serves to
stimulate activity of those tissues and subsequently, expression of
estrus. Alternatively, some metabolic hormone or factor not measured
in this experiment but related to elevated NEFA affects positively the
HPO axis and stimulates expression of estrus. Neither definitive
conclusions nor informed speculations can be formulated for the
effects of NEFA on expression of estrus because there is a paucity of
information relating metabolic hormones and metabolites to
reproductive function in sows. Characterization of metabolic
differences between sows that express estrus promptly after weaning
and those that are delayed in return to estrus is necessary before a

thorough understanding of these data can be realized.

Conclusions
Insulin administered during the postweaning period does not
hasten return to estrus 0f primiparous sows fed maintenance diets.
Insulin, glucose or UN were not different between sows that did and
those that did not express postweaning estrus. Concentration of NEFA
in plasma or some factor related to NEFA appeared to have a positive

effect on expression of postweaning estrus.

 

 

 

 

 

GENERAL DISCUSSION
Many factors have been associated with delayed return to
postweaning estrus in primiparous sows. Previous reports in the
literature have shown that low intake of dietary energy and(or)
protein, loss of body weight and fat, parity of the sow, photoperiod,
season and breed of the sow can influence interval to postweaning
estrus. Many of these factors do not consistently affect interval to

estrus. However, researchers have shown consistently that intake of

 

dietary energy and(or) protein is related negatively to loss of body
weight and fat and to postweaning interval to estrus (Reese et al.,
1982; King and Williams, 1984b; Nelssen et al., 1985; Brendemuhl et
al., 1987). Furthermore, loss of body weight and fat are related
positively to interval to estrus (Reese et al., 1982; King and Dunkin,
1986a). These clinical observations of relationship among dietary
energy and protein intake, loss of body weight and fat and interval to
estrus do not establish cause and effect relationships. Therefore, it

was the aim of this research to identify what factor(s) may or may not

 

be responsible for delayed return to estrus in primiparous sows.

Low intake of feed during lactation causes delayed return to
postweaning estrus (Reese et al., 1982; King and Dunkin, I986a). The
first question to be addressed was: Are the effects of low dietary
intake on interval to estrus mediated through body fat? Several
researchers have hypothesized a minimal threshold of body fatness must
be preserved for females to maintain reproductive functions (Frisch

117

 

 

118

and McArthur, 1974; Kirkwood and Aherne, 1985). No experiments have
been conducted specifically to test this hypothesis in sows. In
Experiment I, primiparous sows were subjected to varying degrees of
negative energy balance during lactation by standardizing suckling
intensity and varying ME intake to produce sows with a wide range of
body fat content at weaning. Thus, the variable of interest was body
fat not diet. If body fat does control postweaning interval to
estrus, one would expect an inverse relationship between body fat at
weaning and interval to estrus. Increasing body fat above the
theoretical minimal threshold would have no effect on interval to
estrus. Neither body fat at weaning nor body fat at first postweaning
estrus explained a large portion (r25.24) of the variation in
postweaning interval to estrus. One may conclude that within the
range of body fatness produced in Experiment I body fat does not
directly control expression of postweaning estrus (Figure 15).
Effects of body fat are confounded with dietary energy intake and
energy balance because diet was used to alter body composition. An
unconfounded test of effects of body fat on interval to estrus would
require alteration of body composition without influencing energy
balance. Methods to alter body composition without affecting energy
balance are not obvious.

Loss of body weight during lactation accounted for a greater
portion of variation in postweaning interval to estrus than did body
fat. Body weight loss of sows in negative energy and nutrient balance
represents loss of fat and muscle tissue (Brendemuhl et al., 1987;

NRC, 1988). Therefore, loss of body protein (muscle tissue) may

 

 

 

 

119

 

 

Low intake of dietary essentials
relative to reproductive needs

/_ i \ e

t? _ _
Low body m, Hormonal and JéLLow bodfl
s

 

 

 

 

 

 

 

 

 

 

 

 

 

 

fat _metaboiic mediator ‘ protein f
\ Stress insulin Glucose NEFA . l
i i ..
' ' Unlikely

\ No 0".“ -£? N0 "g.” + pa way

\ Hypothelamic~pituitary~ /

\ ovarian axis Clinical

/ observation

\ /
it t
[ Expression of estrus J L

 

 

 

Figure 15. Relationships among low intake of dietary essentials,
change in body composition, selected metabolites and incidence
of anestrus.

   

 

120
explain why body weight loss accounts for a greater portion of

variation in postweaning interval to estrus. A direct test of effects
of body protein on interval to estrus in sows must await an accurate
in 111g procedure to quantify body protein or degradation of body
protein in sows.

Metabolizable energy intake during lactation accounted for the
greatest portion (48%) of variation in interval to estrus indicating
that dietary energy intake exerts greater control over postweaning
interval to estrus than either body fat or body weight. Even so, one
must realize that 52% of variation in interval to estrus is still
unexplained. Consideration of results from Experiment 1 permits
formulation of one important conclusion and encourages some
speculation on relationship of dietary energy intake to postweaning
interval to estrus. First, low body fat resulting from restricted
intake of dietary energy does not directly control the postweaning
interval to estrus (Figure 15). From this conclusion, one can
speculate that low body protein does not directly control postweaning
interval to estrus. This supposition awaits definitive testing.
Second, dietary energy intake during lactation is related inversely to
postweaning interval to estrus. As illustrated in Figure 15, dietary
energy may directly influence hormones and(or) metabolites related to
body metabolism which negatively influence HPO function resulting in
delayed return to estrus. Alternatively, some metabolic intermediate
related to loss of body fat and(or) protein may act as the mediator
which negatively influences the HPO axis. Regardless of which pathway
represents the actual situation, some hormonal or metabolic

mediator(s) is implicated in the etiology of delayed return to

 

 

121

 

postweaning estrus. Thus, the remainder of my research was planned to
identify hormonal or metabolic mediators which influence expression of
estrus.

In a preliminary experiment involving daily collection of blood
during the postweaning period, interval to estrus was much shorter
than expected based on results of Experiment I for sows that consumed
7 Mcal ME daily during lactation. Stress can induce expression of
estrus in gilts (Signoret, 1972; Zimmerman, 1976) and ovulation in
ewes (Braden and Moule, 1964). One may hypothesize that stress caused
by physical restraint and sampling of blood had a positive effect on
return to postweaning estrus. Experiment 11 was designed to test
effects of handling-induced stress on expression of postweaning
estrus. Stress induced by daily snaring and sampling of blood did not
affect interval to postweaning estrus. One can conclude that hormonal
and metabolic changes associated with daily episodes of acute stress
do not appear to affect postweaning expression of estrus.

Concentration of insulin in serum is related positively to feed
intake (Cahill, 1971; Armstrong and Britt, 1987). Insulin is
gonadotropic (Harrison and Randel, 1986; Cox et al., 1987) and can
stimulate the pituitary (Adashi et al., 1961). Since serum insulin is
related to feed intake and stimulates HPO axis, insulin may be the
signal which delays or hastens postweaning expression of estrus. The
primary objective of Experiment III was to determine if exogenous
insulin would hasten return to postweaning estrus in sows subjected to
severe restriction of dietary energy during lactation. Exogenous

insulin did not shorten postweaning interval to estrus in the

 

 

 

 

 

 

122
experiment. Interpretation of these data is difficult. Gonadotropic

actions of insulin in gilts are enhanced by liberal intake of dietary
energy (Cox et al., 1987). Sows in this experiment received a limited
amount of feed calculated to satisfy maintenance energy requirements.
Possibly, sows did not receive enough dietary energy during the
postweaning period for insulin to manifest a positive effect.
Alternatively, one may conclude that exogenous insulin has no
favorable effect on expression of postweaning estrus. Further
investigations are required before the latter statement can be
confirmed.

A second objective of Experiment III was to investigate the
relationship among selected blood borne metabolites and expression of
postweaning estrus. Body metabolism adjusts to level of feed intake
and blood borne metabolites reflect the adjustment in metabolism. A
blood borne metabolite may be the signal which controls expression of
estrus. Concentration of glucose and UN were not different between
cyclic and acyclic sows during the postweaning period. Reese et al.
(1984) reported concentration of UN in blood of cyclic and acyclic
sows was not different. Concentrations of metabolites in blood do not
permit evaluation of metabolite turnover. Potentially, turnover of
these metabolites was different between cyclic and acyclic sows and
may be causally related to interval to postweaning estrus.

Concentration of NEFA in plasma was higher throughout the
postweaning period for cyclic compared with acyclic sows. This
difference occurred in the absence of differences in loss of body
weight or backfat. Furthermore, concentration of NEFA in blood is not

related to body fat content in pigs (Wood et al., 1977) or heifers

 

 

 

 

 

 

 

 

 

 

123
(Villa-Godoy, 1987). This would suggest that NEFA have a positive
effect on expression of estrus independent of body fat. No previous
research has reported positive effects of NEFA in blood during the
postweaning period on expression of estrus. Higher concentration of
NEFA in plasma of sows during mid-lactation has been reported for sows
that expressed estrus by d 8 postweaning compared with sows that
expressed estrus after d 23 postweaning (Armstrong et al., 1986).
Non-esterified fatty-acids in blood or some factor related to NEFA
concentration in blood seem to affect positively expression of

postweaning estrus (Figure 15).

 

There is a dearth of information relating metabolic hormones and
metabolites to reproductive function. Characterization of metabolic
differences of sows that display a short interval to estrus compared
with sows that are delayed in their return to estrus should advance
our understanding or at least help focus future questions regarding

reproductive function as it relates to body metabolism.

 

 

 

 

 

SUMMARY AND CONCLUSIONS

Intake of ME during lactation is related negatively to loss

of body weight and backfat depth. Body fat of sows at weaning did not

 

account for a significant portion of the variation in postweaning
interval to estrus. Similarly, body fat of sows at first postweaning

estrus was not related to interval from weaning to estrus. When all

 

factors measured in Experiment I are considered, intake of ME during
lactation accounted for the greatest portion of variation in interval 1
from weaning to estrus.

Restriction of dietary ME intake during lactation to 58% of
recommended levels consistently delayed return to postweaning estrus.
Stress associated with daily snaring and sampling of blood during the
postweaning period had no effect on interval from weaning to estrus.
Hormonal and metabolic changes associated with acute stress could not

ameliorate the detrimental effects of dietary restriction during

 

lactation on expression of postweaning estrus.

Insulin administered once daily during the postweaning period
increased concentation of insulin in serum and depressed concentration
of glucose in plasma throughout the 24 h period after injection.
Exogenous insulin tended to increase mean concentration of NEFA and
decrease mean concentration of UN in plasma during the postweaning
period. Exogenous insulin did not hasten return to estrus in

primiparous sows consuming diets designed to satisfy energy and
124

 

 

125

nutrient requirements for maintenance.

There were no differences in concentration of insulin, glucose or
UN in blood at weaning, at end of the experiment or throughout the
postweaning period between sows that displayed estrus and sows that
were anestrous. In contrast, concentration of NEFA in plasma at
weaning and throughout the postweaning period was associated
positively to expression of postweaning estrus. Loss of body weight
and backfat depth and litter weight at weaning were similar between
cyclic and acyclic sows.

The first conclusion is that neither body fat at weaning nor body
fat at first estrus plays a primary role in controlling expression of
postweaning estrus in primiparous sows. Timing of postweaning estrus
is not dependent on a minimal threshold of body fat. Effects of ME
intake during lactation on interval to estrus are more pronounced than
effects of body fat. Therefore, some factor related to ME intake but
independent of body fat may control expression of postweaning estrus.

The second conclusion is that the stress associated with daily
physical restraint and sampling of blood under the conditions of
Experiment 11 does not influence expression of postweaning estrus.

Finally, insulin administered during the postweaning period does
not hasten return to estrus of primiparous sows fed maintenance diets.
Concentration of NEFA in plasma or some factor related to NEFA appears

to have a positive effect on expression of postweaning estrus in

primiparous sows.

 

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