Conceptus development in intact and unilaterally hysterectomized-ovariectomized gilts

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Title:
Conceptus development in intact and unilaterally hysterectomized-ovariectomized gilts interrelationships between hormonal status, placental development, fetal fluids and fetal growth
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xiii, 158 leaves : ill. ; 28 cm.
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English
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Knight, James William, 1948-
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Subjects / Keywords:
Swine -- Embryos   ( lcsh )
Swine -- Growth   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Includes bibliographical references (leaves 146-157).
Statement of Responsibility:
by James William Knight.
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Typescript.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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notis - ABZ3806
oclc - 02274189
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AA00003529:00001

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CONCEPTS DEVELOPMENT IN INTACT AND UNILATERALLY
HIYSTERECTOMIZED-OVARIECTOMIZED GILTS:
INTERRELATIONSHIPS BETWEEN HORIMIONAL STATUS,
PLACENTAL DEVELOPMENT, FETAL FLUIDS AND FETAL GROWTH







By



JAMES WILLIAM KNIGHT


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA

1975















AC KNOWLE DGMENTS


The author wishes to express his sincere appreciation

and gratitude to the members of his supervisory committee,

Dr. Fuller W. Bazer, chairman, Dr. Donald Caton, Dr. William

W. Thatcher and Dr. A. C. Warnick, for their advice and assis-

tance in this research. The assistance and invaluable guid-

ance of Dr. Donald E. Franke in conducting the statistical

analyses of the data in this study is also gratefully acknowl-

edged.

Special appreciation is expressed to Dr. Fuller W. Bazer

for his invaluable and always willing assistance in all

phases of this study from formulation to completion. Without

his assistance, direction, guidance and encouragement this

research would not have been possible. The author considers

it a great privilege to have worked with and learned from

this excellent researcher.

The aid and advice of Dr. W. W. Thatcher and Dr. P. S.

Kalra in conducting the hormone assay procedures in this

study are also greatly appreciated. Special thanks are also

due to Dr. II. I). Wallace, who kindly furnished the gilts used

in this study. Thanks are also expressed to Dennis L.

Rodebusch for care and feeding of the animals and his assis-

tance with breeding and sur--.cry.










The generous surgical and data collection assistance

of fellow graduate students Thomas T. Chen and Albert C.

Mills III is also gratefully acknowledged. The completion

of this research would not have been possible without the

technical aid generously and enthusiastically offered by

Norma J. Baldwin, Marilyn Frank, Elizabeth Patterson and

Linda J. Owens. The invaluable assistance of these ladies

in one or more phases of this study is gratefully acknowledged

and sincerely appreciated.

The author also wishes to acknowledge the support and

encouragement of his mother, Mrs. Theda F. Baum, and the

guidance and inspiration of his late father, Mr. L. C. Knight.

Finally, the author wishes to express his deep appreciation

for the sacrifices, constant encouragement, unceasing aid and

love generously given by his wife, Sharon Ann, and it is to

her that this effort is dedicated.
















TABLE OF CONTENTS


Page


ACKNOWLEDGE ENTS . .

LIST OF TABLES . .

LIST OF FIGURES . .

ABSTRACT . .

INTRODUCTION .....

CHAPTER

I REVIEW OF LITERATURE .


General Aspects of Embryonic Mortality
Uterine Protein Secretions .
Porcine Conceptus Development .
Progesterone and Estrogen Levels in Pigs

II CONCEPTS DEVELOPMENT IN INTACT AND
UN I LATERALLY IIYSTERECTOMIZED-OVARIECTOMIZED
GILTS: INTERRELATIONSHIPS BETWEEN HORMONAL
STATUS, PLACENTAL DEVELOPMENT, FETAL
FLUIDS AND FETAL GROWTH . .

Materials and Methods . .
Results and Discussion . .

GENERAL DISCUSSION . .

SUMMARY . . ..

LIST OF REFERENCES -


4
. 13
. 25
S. 37


S 48
. 60

. 125

S. 139

. 146


BIOGRAPHICAL SKETCH


xi


. 158
















LIST OF TABLES


Table


1 Validation of Estrone Assay ... 58

2 Analysis of Variance Expected Mean Squares
for Fetal Data . . 59

3 Comparison of Number of Corpora Lutea and
Number of Live Embryos in Intact Control
and Unilaterally Ilysterectomized-Ovariec-
tomized Gilts at Various Stages of Gestation .61

4 Comparison of Number of Dead Embryos and
Percent Fetal Survival in Intact Control
and Unilaterally IIysterectomized-Ovariec-
tomized Gilts at Various Stages of Gestation 62

5 Comparison of Empty Uterine Weight and
Length of the Uterine Horn(s) in Intact
Control and Unilaterally Ilysterectomized-
Ovariectomized Gilts at Various Stages of
Gestation . . 63

6 Simple Correlation Coefficients Between
Various Selected General and Conceptus
Measurements . . 65

7 Simple Correlation Coefficients Between
Uterine Surface Area, Uterine Surface Area
per Fetus, Empty Uterine Weight and the
Various Placental and Fetal Measurements 66

8 Comparison of Uterine Surface Area and
Uterine Surface Area per Fetus in Intact
Control and Unilaterally Ilysterectomized-
Ovariectomized Gilts at Various Stages of
Gestation . . 68

9 Comparison of Radial Vein Plasma Protein
Concentration and Uterine Vein Plasma
Protein Concentration in Intact Control
and Unilaterally lHysterectomized-
Ovariectomized Gilts at Various Stages
of Gestation . . 69


Page











LIST OF TABLES (continued)


10 Comparison of Uterine Artery Plasma Protein
Concentration and Umbilical Vessel Plasma
Protein Concentration in Intact Control and
Unilaterally lyste rectomiized-Ovariectomized
Gilts at Various Stages of Gestation .. .70


11 Comparison of Allantoic Fluid Volume in
Intact Control and Unilaterally Ilysterec-
tomized-Ovariectomized Gilts at Various
Stages of Gestation . .

12 Comparison of Allantoic Fluid Protein Con-
centration and Allantoic Fluid Total Protein
in Intact Control and Unilaterally lIysterec-
tomized-Ovariectomized Gilts at Various
Stages of Gestation . .


. 71





. 74


13 Comparison of Amniotic Fluid Volume in
Intact Control and Unilaterally Ilysterec-
tomized-Ovariectomized Gilts at Various
Stages of Gestation . .. 76

14 Comparison of Amniotic Fluid Protein Con-
centration and Amniotic Fluid Total Protein
in Intact Control and Unilaterally llysterec-
tomized-Ovariectomized Gilts at Various
Stages of Gestation . .. 78

15 Comparison of Placental Length in Intact
Control and Unilaterally Hysterectomized-
Ovariectomized Gilts at Various Stages of
Gestation . . 79

16 Comparison of Placental Weight of Intact
Control and Unilaterally Ilysterectomized-
Ovariectomized Gilts at Various Stages of
Gestation . . 81

17 Comparison of Placental Surface Area in
Intact Control and Unilaterally Ilysterec-
tomized-Ovariectomized Gilts at Various
Stages of Gestation . 83

18 Comparison of Placental Displacement
Volume in Intact Control and Unilaterally
flysterectomized-Ovariectomized Gilts at
Various Stages of (;station . 84


Table


Page











LIST OF TABLES (continued)


Table Page

19 Simple Correlation Coefficients Between
Placental and Fetal Measurements ... .85

20 Comparison of Total Number of Areolae,
Interior Sections,and Total Number of
Areolae, Polar Sections, in Intact Control
and Unilaterally Ilysterectomized-Ovariec-
tomized Gilts at Various Stages of Gestation 86

21 Comparison of Areolae Surface Area, Interior
Sections, and Areolae Surface Area, Polar
Sections, in Intact Control and Unilaterally
llysterectomized-Ovariectomized Gilts at
Various Stages of Gestation . 87

22 Comparison of Total Number of Areolae per
Placenta and Total Areolae Surface Area per
Placenta in Intact Control and Unilaterally
Ilysterectomized-Ovariectomized Gilts at
Various Stages of Gestation .. 88

23 Comparison of Fetal Crown-Rump Length in
Intact Control and Unilaterally llysterec-
tomized-Ovariectomized Gilts at Various
Stages of Gestation . 90

24 Comparison of Fetal Wet Weight in Intact
Control and Unilaterally H1ysterectomized-
Ovariectomized Gilts at Various Stages of
Gestation . . 91

25 Comparison of Fetal Dry Weight and Percent
Fetal Moisture in Intact Control and
Unilaterally HIysterectomized-Ovariectomized
Gilts at Various Stages of Gestation .. 92

26 Prediction Models for Fetal Wet Weight .. 95

27 Comparison of Radial Vein Progestin Con-
centration and Uterine Vein Progestin
Concentration in Intact Control and
Unilaterally Hysterectomized-Ovariectomi zed
Gilts at Various Stages of Gestation .. 96

28 Comparison of Uterine Artery Progestin
Concentration and UIrhilical Vessel Proges-
tin Concentration in Intact Control and
Unilaterally flysterectomized-Uvariectomized
Gilts at Various Stages of Gestation .. 98











LIST OF TABLES (continued)


Table Page

29 Comparison of the Radial Vein minus
Uterine Vein Progestin Difference in
Intact Control and Unilaterally Hysterec-
tomized-Ovariectomized Gilts at Various
Stages of Gestation .. 99

30 Simple Correlation Coefficients Between
Steroid Hormone Concentrations and Various
Selected General and Conceptus Responses 102

31 Comparison of Allantoic Fluid Progestin
Concentration and Total Allantoic Fluid
Progestins in Intact Control and Unilater-
ally IHysterectomized-Ovariectomized Gilts
at Various Stages of Gestation .. 103

32 Comparison of Amniotic Fluid Progestin
Concentration and Total Amniotic Fluid
Progestins in Intact Control and Unilater-
ally Ilysterectomized-Ovariectomized Gilts
at Various Stages of Gestation ... 104

33 Simple Correlation Coefficients Between
the Estrogens from the Various Sources .. 106

34 Simple Correlation Coefficients Between
Plasma Progestins and Estrogens .. .108

35 Comparison of Radial Vein Estrone Concen-
tration and Uterine Vein Estrone Concen-
tration in Intact Control and Unilaterally
Hysterectomized-Ovariectomized Gilts at
Various Stages of Gestation . 109

36 Comparison of Radial Vein Estradiol Concen-
tration and Uterine Vein Estradiol Concen-
tration in Intact Control and Unilaterally
Hysterectomized-Ovariectomized Gilts at
Various Stages of Gestation . 110

37 Comparison of Uterine Artery Estrone Con-
centration and Umbilical Vessel Estrone
Concentration in Intact Control and
Unilaterally Hysterectomi zed-Ovariectomized
Gilts at Various Stages of Gestation .. .111


viii











LIST OF TABLES (continued)


38 Comparison of Uterine Artery Estradiol Con-
centration and Umbilical Vessel Estradiol
Concentration in Intact Control and Unilater-
ally Hysterectomized-Ovariectomized Gilts
at Various Stages of Gestation . 112


39 Comparison of Allantoic Fluid Estrone Con-
centration and Total Allantoic Fluid Estrone
in Intact Control and Unilaterally Hysterec-
tomized-Ovariectomized Gilts at Various
Stages of Gestation . .

40 Comparison of Allantoic Fluid Estradiol Con-
centration and Total Allantoic Fluid Estradiol
in Intact Control and Unilaterally Iysterec-
tomized-Ovariectomized Gilts at Various
Stages of Gestation . .

41 Comparison of Amniotic Fluid Estrone Con-
centration and Total Amniotic Fluid Estrone
in Intact Control and Unilaterally lysterec-
tomized-Ovariectomized Gilts at Various
Stages of Gestation . .

42 Comparison of Amniotic Fluid Estradiol Con-
centration and Total Amniotic Fluid Estradiol
in Intact Control and Unilaterally Hysterec-
tomized-Ovariectomized Gilts at Various
Stages of Gestation . .


. 114





. 115





. 122


. 123


Table


Page
















LIST OF FIGURES


Figure Page

1 Interrelationships between allantoic fluid
estrone concentration, allantoic fluid
volume, allantoic fluid total protein and
placental length in intact control gilts
from day 20 to day 100 of gestation .. 118










Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of )octor of Philosophy


CONCEPTS DEVELOPMENT IN INTACT AND UNILATERALLY
IIYSTERECTOMI ZED-OVARIECITOMI1ZED GILTS:
INTERRELATIONSHIPS BETWEEN HORMONAL STATUS,
PLACENTAL DEVELOPMENT, FETAL FLUIDS AND FETAL GROWTH

By

James William Knight

June, 1975

Chairman: Fuller W. Bazer
Major Department: Animal Science


Conceptus (placental membranes, fetal fluids and fetus)

development in intact control (IC) and unilaterally hysterec-

tomized-ovariectomized (UHOX) gilts was characterized between

days 20 and 100 of gestation to evaluate the effects of intra-

uterine crowding on concepts development. Results indicated

that the number of live and dead embryos, percent fetal sur-

vival, fetal crown-rump length and fetal weight were not sig-

nificantly (P>.10) different prior to day 35 of gestation in

the IC and UHIOX gilts; however, all placental measurements

were significantly (P<.01) greater in the IC gilts at all

stages of gestation. All lines of evidence in this study

emphasized the importance of developing adequate placental

mass early in gestation and suggested that placental insuffi-

ciency was the primary cause of increased fetal death and

decreased fetal growth which occurred after day 35 of gesta-

tion in the UHOX gilts. These data indicated that limited

placental mass was capably of supporting the relatively










limited fetal growth which occurred prior to day 35 of gesta-

tion. However, this placental insufficiency resulted in a

significant (P<.01) decrease in fetal growth rate and in-

creased fetal mortality in UHOX gilts as gestation progressed.

Fetal growth was significantly (P<.01) correlated with pla-

cental development.

Progestins, estrone and estradiol concentrations in the

radial vein, uterine vein, uterine artery, umbilical vessels,

allantoic fluid and amniotic fluid were characterized. Data

indicated that steroid hormone changes and changes in con-

ceptus development were interrelated. Plasma progestins were

relatively constant between days 20 and 80 of gestation, but

decreased precipitously after day 80. Estrone concentration

was significantly (P<.01) greater than estradiol concentra-

tion in all sources and at all stages of gestation, but their

relative patterns of change were identical. The most dramatic

and marked changes in estrogen concentrations were found in

allantoic fluid. Allantoic fluid estrogens increased rapidly

from day 20 to 30, decreased to day 40, remained relatively

constant between days 40 and 60 and increased rapidly from

days 60 to 100 of gestation.

The data suggest that the rapid increase in allantoic

fluid and circulating plasma estrogens and the increasing

osmotic gradient due to protein movement into the allantoic

fluid from the uterine glands, provided the primary stimuli

for the rapid increase in allantoic fluid volume between days

20 and 30 of gestation. iThls early increase in allantoic


xii










fluid volume appeared to be essential for expansion of the

chorio-allantois membranes, which forced those membranes into

apposition with the uterine glands, thus allowing formation

of the maximum number of placental areolae. The placental

areolae have been established as the site of absorption and

transport of uterine gland secretions. The data suggest that

days 20 to 30 represent the most critical stage of gestation

since the extent of placental development which occurs during

this period ultimately regulates fetal growth, fetal survival

and perhaps even postnatal survival.

This study indicates that future experiments concerned

with fetal growth and survival must consider methods for

stimulating maximum placental development early in gestation

if placental insufficiency and its resulting undesirable

effects on fetal development are to be overcome.


xiii















INTROI)UCTION


Numerous reports in the literature indicate that litter

size in swine at 25 to 40 days of gestation can be increased

by hormonal superovulation with pregnant mares' serum gonado-

trophin (P;ISG) (Gibson et al., 1963; Longenecker et al., 1965;

Hunter, 1966; Day et al., 1967; Longenecker and Day, 1968; and

Christenson et al., 1970) or by embryo superinduction (Bazer

et al., 1969a,b; Fenton et al., 1969a). However, it has also

been well established that superovulation with PMSG (Hammond,

1921; Perry, 1954; Rathnasabapathy et al., 1956; King and

Young, 1957; Hunter, 1966; Dziuk, 1968; Longenecker and Day,

1968; Pope et al., 1968; and Bazer et al., 1969c) and embryo

superinduction (Bazer et al., 1969a,b; Fenton et al., 1969b)

result in increased embryonic mortality so that litter size

at term is similar for control and treated gilts.

Likewise, litter size and embryonic survival have been

demonstrated to be similar for control and unilaterally

hysterectomized-ovariectomized (UIOX) gilts up to day 30 of

gestation (Dziuk, 1968; Fenton et al.,1970; and Webel and

Dziuk, 1974). However, after day 30, the one-half reduction

in uterine endometrial surface area in the UIOX gilts has

been shown to result in increased embryonic mortality (Dziuk,

1968; Fenton et al., 1970; ;ad Webel and Dziuk, 1974) so that







2


litter size at term in UIIOX gilts was only one-half that for

control gilts (Fenton et al., 1970).

Although several workers have suggested that an inhibi-

tion of placental development due to intra-uterine crowding

may be the cause of increased fetal mortality in the previously

mentioned studies, few studies have included measurements of

placental parameters and none have characterized concepts

development under normal and "crowded" conditions in a manner

which would allow a definitive conclusion as to the cause of

fetal mortality following intra-uterine crowding.

Warwick (1928) reported, and Waldorf et al. (1957) and

Pomeroy (1960) confirmed, that there was a direct and highly

significant correlation between placental size and fetal size

in gilts. Eckstein and McKeown (1955) and Eckstein et al.

(1955) made a similar observation in guinea pigs. Rathnasa-

bapathy et al. (1956) demonstrated highly significant (P<.01)

correlations between fetal weight and uterine surface area

per fetus and between fetal survival and uterine surface area

per fetus in gilts at day 55 of gestation.

While plasma progestin, estrone and estradiol changes

in the porcine have been described early and late in gesta-

tion, there has been only one known characterization of their

concentrations throughout gestation (Robertson and King, 1974).

There have been no known reports which relate the changes in

steroid hormone concentrations to changes in concepts devel-

opment.








3


This study was undertaken to characterize porcine con-

ceptus (placental membranes, fetal fluids and fetus) develop-

ment during gestation under normal conditions and conditions

of intra-uterine crowding resulting from unilateral hysterec-

tomy-ovariectomy. This experimental design allowed for com-

parison of the patterns of concepts development under these

two treatment regimes. The determination of specific effects

of intra-uterine crowding on concepts development would,

therefore, provide an explanation for the increased incidence

of fetal mortality following intra-uterine crowding. This

study was undertaken not just to characterize concepts devel-

opment per se, but rather to examine the interrelationships

and/or physiological significance of the changes which occur

in concepts development during gestation. Additionally, the

circulating plasma and fetal fluid levels of progestins,

estrone and estradiol were analyzed in order to more fully

characterize their changes over the course of gestation and

specifically to relate changes in these steroid hormone con-

centrations to various aspects of concepts development.















CHAPTER I

REVIEW OF LITERATURE



General Aspects of Embryonic Mortality


Embryonic mortality in swine has been reported to account

for a loss of 30 to 50 percent of potential offspring (Bram-

bell, 1948; Casida, 1953; Squires et al., 1952; Perry, 1954;

Rathnasabapathy et al., 1956; and Lerner et al., 1958).

Embryonic mortality or prenatal death loss refers to the dif-

ference between ovulation rate and litter size at a specified

stage of gestation. Estimates of embryonic mortality are

based upon the assumptions that the number of functional cor-

pora lutea (CL) represent the number of ova shed and, there-

fore, the number of potential embryos and that the difference

between the number of CL and the number of live embryos repre-

sents embryonic mortality.

Ovulation rate, which obviously sets the upper limit, is

the first factor which could affect litter size. Since swine

have multiple ovulations and since each CL is seldom repre-

sented by a live pig at term, ovulation rate can generally be

discounted as a major factor limiting litter size. It is

well documented that ovulation rate in swine can be increased

by the administration of pregnant mare serum gonadatrophin

(PMSG); however, superovulati on has not been shown to







5


significantly increase litter size at term (Gibson et al.,

1963; Hunter, 1964, 1966; Longenecker et al., 1965; Day et al.,

1967; Longenecker and Day, 1968; and Pope et al., 1968).

It has been well established in swine that under normal

conditions, with adequate quantities of viable spermatozoa

present in the semen and proper time of breeding, essentially

100 percent of the ova shed will be fertilized (Hammond, 1921;

Squires et al., 1952; Perry, 1954; and Baker et al., 1958).

Boyd (1965) reported fertilization failure in swine to be

essentially an all or none phenomenon that is considered

negligible under most conditions. Also, within the physio-

logical range, there is no evidence to suggest that either an

increase or a decrease in ovulation rate has any effect on

fertilization rate (Wrathall, 1971).. Consequently, fertili-

zation failure can be ruled out as a major factor responsible

for reducing litter size in swine.

Since available data indicate that the reproductive rate

in swine is not generally limited due to either an inadequate

ovulation rate or fertilization failure, the major factor

affecting the reproductive rate must be the embryonic death

loss which occurs between fertilization and parturition.

Much research has been conducted in an attempt to determine

when during gestation embryonic mortality occurs, factors

responsible for or contributing to its occurrence and methods

to control the intrinsic factors which appear to regulate

embryonic mortality.










Numerous workers have established that the major portion

of embryonic mortality in swine occurs prior to day 25 of

gestation (Hammond, 1921; Corner, 1923; Brambell, 1948;

Squires et al., 1952; Haines et al., 1958; Baker et al., 1956;

Lerner et al., 1958; and IIanly, 1961). Lerner et al. (1958)

reported that two-thirds of the embryonic mortality occurring

in swine by day 25 had occurred prior to day 17 of gestation.

A portion of this embryonic mortality may be due to in-

herent genetic defects or chromosomal abnormalities, maternal

limitations such as an inadequate uterine circulatory system,

or a low level of some essential biochemical factor necessary

for embryonic development which may cause only a portion of

the litter to die (Runner, 1951). Hammond (1914) suggested

that a portion of embryonic mortality is due to the inability

of runts, i.e., smaller pigs, to compete in the struggle for

some nutritional factors) provided by the uterus. Later

studies suggested that the inability of these runts to ade-

quately compete may be due to limited placental surface area

(Warwick, 1928; Waldorf et al., 1958; Pomeroy, 1960).

Whole litter losses in swine, when they occur, generally

occur between fertilization and the 12th day of pregnancy.

The loss occurring during this period has been studied by

Perry and Rowlands (1962). They found that prior to day 6 all

fertilized eggs appeared to be developing normally; between

day 6 and day 9 (when blastocyst formation begins and the

zona pellucida is lost), degenerating eggs were rather common

and accounted for 22 percent of the total fertilized eggs.










They also found an even greater loss (28 percent) in gilts

and sows killed between 13 and 18 days of pregnancy. Corner

(1923) also found a high incidence of visible abnormalities

in embryos recovered between days 6 and 10 of pregnancy.

Laing (1968), in a study of embryo losses between days 10 and

25 of gestation, confirmed that most embryonic mortality had

occurred by day 13 of pregnancy.

Reports of whole litter losses in swine during the latter

stages of gestation are rare, suggesting that the incidence

of such losses is very low. The largest available estimate

of whole litter losses in late gestation is 5 percent (Wilson

et al., 1949). This figure was based on a study involving

gilts and sows with a history of reproductive problems and

is probably higher than would be expected with a random popu-

lation of females.

The occurrence of intra-uterine migration of embryos in

swine has been well established (Corner, 1923; Warwick, 1926;

and Kelly, 1928). Perry and Rowlands (1962) found that 75

percent of embryonic deaths in swine occurred between days 6

and 9 of pregnancy when intra-uterine spacing was taking

place. Lasley et al. (1963) examined the frequency of intra-

uterine migration and its relationship to embryonic mortality.

The frequency of intra-uterine migration, as determined by

the number of embryos in any one uterine horn exceeding the

number of CL on the adjacent ovary, was 41.2 percent. Despite

unequal ova production by the ovaries, the embryos were found

to be equally divided between the uterine horns at day 55 of










gestation. When intra-uterine migration was observed, litters

contained 1.24 more fetuses. This suggested that intra-

uterine migration may be important in limiting embryonic

death losses. Dhindsa and Dziuk (1965) reported that embryo

migration occurred between days 8 and 10 of pregnancy in

swine. Waite and Day (1967) conducted an investigation to

determine the time of intra-uterine migration by using a pro-

cedure of depositing semen directly into one uterine horn to

obtain unilateral fertilization. In their study, intra-

uterine migration was first observed on day 11 of pregnancy.

It is worthy of note that elongation of the trophoblast was

not observed before day 12 of gestation, therefore embryo

migration took place before extensive elongation of the tro-

phoblast.

Extensive work has been done to demonstrate that super-

ovulation of swine with PMSG results in increased embryonic

death rates so that litter size at farrowing is not signifi-

cantly different between superovulated and control gilts or

sows (Hammond, 1921; Perry, 1954; Rathnasabapathy et al.,

1956; King and Young, 1957; Hunter, 1966; Dziuk, 1968;

Longenecker and Day, 1968; Pope et al., 1968; and Bazer et al.,

1969c). Similar findings have been observed in experiments

involving superovulation of other polytocous species. Adams

(1960) showed that the effect of superovulation in rabbits

was to actually decrease the number of fetuses surviving to

term. Hafez (1964) also found that superovulation failed to

increase litter size in rabbits and that the maximum number










of fetuses at term was 15 per litter and 8 per uterine horn.

In mice, Fowler and Edwards (1957) reported that superovula-

tion did not increase litter size and suggested that intra-

uterine crowding may be responsible for abnormal embryos.

However, McLaren and Mlichie (1956) reported an increase in

litter size in mice following superovulation, although per-

cent embryonic mortality was increased over control levels.

Despite the aforementioned data which indicate that

superovulation of gilts and sows does not result in increased

litter size at term, there are numerous reports which indicate

that litter size is greater at 25 to 40 days of gestation for

superovulated as compared to control gilts and sows (Gibson

et al., 1963; Longenecker et al., 1965; Hunter, 1966; Day

et al., 1967; Longenecker and Day, 1968; Bazer et al., 1969c;

and Christenson et al., 1970).

Spalding et al. (1955) reported that ova from induced

and normal ovulations were equally fertilizable and that they

reached the same stage of maturation. Hunter (1966) found

15.8 percent of the eggs from superovulated gilts to be either

polyspermic or fragmenting. These abnormalities were particu-

larly evident when the ovulation rate exceeded 25. Much

lower percentages of abnormal ova following superovulation

have been reported by Hancock (1961) who found only 0.2 per-

cent to be abnormal, and Polge and Dziuk (1965) who found

2.0 percent of the ova to be abnormal. These data suggested

that only a small amount of the embryonic loss in superovu-

lated gilts may be attributl: ile to abnormal ova. The fact










that there was a significant increase in litter size at 25

days of gestation, but not at term following superovulation

of gilts and sows suggested that some additional factors)

were also acting after 25 days of gestation to limit litter

size. The most obvious factor which may limit fetal growth

and survival is placental mass. Intra-uterine crowding

appears to inhibit placental development which occurs during

the first 60 days of gestation (Warwick, 1928; Pomeroy, 1960).

Hunter (1966) reported that increased litter size in super-

ovulated gilts was associated with an overlap of placental

membranes and a reduction in placental weight and allantoic

fluid volume. He found that these factors did not affect

embryonic development at day 25 of gestation. However, it

has been shown that placental size and fetal size are highly

correlated in the latter stages of pregnancy in swine (War-

wick, 1928; Waldorf et al., 1958; and Pomeroy, 1960).

Experiments involving embryo superinduction have provided

similar results to those involving hormonal superovulation.

Bazer et al. (1969a) reported that average litter size at 90

days of gestation in gilts was similar for unoperated controls,

sham operated controls, and gilts subjected to three levels of

embryo superinduction. The number of CL were counted for

each recipient and considered to represent the number of

"native" embryos. Superinduction was achieved in each of the

three groups by transferring 2- to 4-cell embryos into the

uteri of pregnant gilts to increase the number of potential

embryos to either 16, 22 or 28. Upon slaughter at










approximately 90 days of gestation average litter size in the

five groups was 9.9, 8.2, 8.8, 8.5 and 9.9, respectively.

The authors referred to the uterine mechanism which acted to

limit litter size to a level characteristic of the gilts

being studied as "uterine capacity." In a further study,

Bazer et al. (1969b) compared embryonic survival at 25 and

105 days of gestation following embryo superinduction. In

the group of gilts slaughtered at 25 days of gestation, the

number of potential embryos (CL plus transferred embryos) was

an average of 23.9 and the average litter size was 12.0 (50

percent survival). At 105 days of gestation there was an

average of 23.6 potential embryos and an average litter size

of 9.8 (42 percent survival). These data further indicated

that the greatest reduction in litter size occurred prior to

25 days of gestation, but also suggested that a second factor

may be acting later in gestation. Perhaps extensive intra-

uterine crowding may have occurred early in gestation and led

to reduced placental development and, therefore, a further

reduction in litter size.

Fenton et al. (1969a) reported that embryo superinduction

of gilts with 7-day blastocysts resulted in a significant

increase in average litter size at day 25 of pregnancy. In

contrast to these results, Fenton et al. (1969b), using 2.5-

day and 7-day embryos, and Schwartz (1970), using 7-day

embryos, reported that average litter size at 25 days of

gestation was not significantly different from that of con-

trol gilts.










Ovarian compensation in terms of ovulation rate follow-

ing the surgical removal of one ovary has been demonstrated

in swine. Compensation by the remaining ovary of unilater-

ally ovariectomized gilts results in approximately the same

number of ovulations from one ovary as the total from both

ovaries of an intact gilt (Burrows, 1949; Brinkley et al.,

1964; Fenton et al., 1970; and Knight et al., 1973a).

The phenomenon of compensatory ovulation following uni-

lateral hysterectomy-ovariectomy (UIIOX) has been utilized to

study the effects of intra-uterine crowding and the restric-

tion of uterine endometrial surface area on embryonic mortal-

ity. In swine, Dziuk (1968), Fenton et al. (1970) and Webel

and Dziuk (1974) have reported that UIIOX treatment does not

affect litter size up to day 30 of pregnancy. Dziuk (1968)

ligated uterine horns so that embryos on one side of the

uterus had only one-half the uterine space available to

embryos in the contralateral uterine horn. He found that

varying the amount of uterine space available per embryo had

no effect on embryonic survival in swine up to day 25 of ges-

tation. However, he did suggest that intra-uterine crowding

might become increasingly important at later stages of ges-

tation and that embryos at early stages of gestation, due to

their small size, may obtain adequate nutrition from less

than normal placental surface area. Unfortunately, placental

parameters were not measured in his study.

Fenton et al. (1970) reported that although the quantity

of uterine tissue did not limit litter size at 25 days of










gestation, litter size at 105 days of gestation was decreased

by approximately one-half in gilts which were unilaterally

hysterectomized-ovariectomized. Similarly, Webel and Dziuk

(1974) found that increasing or decreasing the amount of

uterine space available per embryo over the normal amount

available did not significantly affect embryonic survival up

to 30 days of gestation. However, after day 30 when uterine

space was reduced to one-half that of untreated females, the

proportion of fetuses surviving was reduced below control

levels. These data suggest the possibility that intra-uterine

crowding inhibited placental development during the first 30

days of pregnancy, i.e., the period during which rapid placen-

tal growth occurs in swine (Warwick, 1928; Pomeroy, 1960).

This results in placental insufficiency which may then limit

fetal growth and survival in the latter stages of gestation.



Uterine Protein Secretions


It was suggested by Iammond (1914, 1921) that some

factors) secreted by the uterus played a role in controlling

litter size. It has been well established that embryos of

swine (Murray et al., 1971), mice (Kirby, 1962; Orsini and

McLaren, 1967), rabbits (Pincus and Kirsh, 1936; Adams, 1958),

rats (Alden, 1942) and sheep (Wintenberger-Torres, 1956) must

reach the uterine environment for continued development beyond

the blastocyst stage, suggesting that some biochemical factor

in the uterine lumen was essential for continued embryonic

growth.










Histological changes in the uterine endometrial epithe-

lium have been well described and documented. Corner (1921)

reported that proliferation of the uterine endometrial epithe-

lium of the sow began on day 8 of the estrous cycle or preg-

nancy. lie described an enlargement both in height and diame-

ter of the uterine epithelial cells. By day 10 of the estrous

cycle and pregnancy, the uterine epithelial cells were marked

by cytoplasmic protrusions on the free surface of the cells.

It was suggested that this phase was of physiological sig-

nificance relative to migration and spacing of embryos and

implantation.

The secretions of the endometrium and uterine glands were

described by Bonnet (1882) and Grosser (1927) as "uterine

milk." It was described as being secreted onto the surface

of the uterine mucosa where it was then absorbed by the chori-

onic epithelium of the placenta. In more recent years, uter-

ine luminal fluids of several species have been studied.

These fluids have been found to consist primarily of proteins.

Junge and Blandau (1958) found four major electrophoretic

components present at low levels in rat uterine fluid. Ring-

ler (1961) reported that rat uterine fluids contained pro-

teins with five electrophoretic components of which only one,

a pre-albumin fraction, was specific to uterine fluid. The

other four proteins were thought to be an ultrafiltrate of

plasma. Albers and Castro (1961), using Ouchterlony gel dif-

fusion and immunoelectrophoresis, found rat uterine fluid to

contain five protein components. They also concluded that










only one of the five components was specific to uterine fluid.

The electrophoretic mobility of this uterine specific com-

ponent was similar to serum beta-globulins. They suggested

that this protein was the result of active secretion by the

endometrium. Its presence was dependent upon the hormonal

status of the animal rather than a result of simple filtra-

tion from the serum.

Stevens et al. (1964) used three techniques to character-

ize the proteins from ligated uteri of rabbits. Using diffu-

sion-in-gel, 13 antigenic components, three of which were

unique to uterine fluid, were found. Eight components were

revealed by moving boundary electrophoresis; two of which

were found to be specific to uterine fluid. Immunoelectro-

phoresis showed five antigens specific to uterine fluid.

Further electrophoretic studies indicated that two of the

five antigens migrated to the pre-albumin region and three

migrated similar to beta-globulins.

Krishnan and Daniel (1967) isolated a uterine specific

protein, which they termed "blastokinin," from the rabbit

uterus during the period of blastulation and blastocyst

development. Blastokinin first appeared on day 3 post coitum

(p.c.), reached its maximum concentration on day 5 p.c. and

progressively declined until day 9 p.c. This protein frac-

tion was also observed in the uterine fluid from pseudopreg-

nant rabbits on day 7 p.c. and in blastocoelic fluid of day

6 blastocysts. It was not present in uterine fluid accumu-

lated by ligation of the uit I-us from day 3 to day 10 of










pregnancy, maternal or fetal serum, or fetal amniotic fluid

collected on day 24 of pregnancy. The molecular weight of

blastokinin was first estimated by Sephadex G-200 gel filtra-

tion to be 27,000, and it was found to be a glycoprotein.

Later analyses of blastokinin (Krishnan and Daniel, 1968)

showed that amino acids constituted approximately 74 percent

of the protein and that carbohydrates make up 6 percent. A

corrected estimate of the molecular weight of blastokinin

was later reported by Murray et al. (1972b). Utilizing tech-

niques of gel filtration, equilibrium ultracentrifugation and

Sodium Dodecyl Sulfate (SDS) electrophoresis, the molecular

weight estimate of blastokinin was 15,000. They further

suggested that blastokinin was a globular protein which was

composed of subunits.

Beier (1968) isolated the same uterine specific protein

from rabbits and termed it "uteroglobin." He initially esti-

mated the molecular weight to be approximately 30,000 and

reported that it constituted approximately 22 percent of

normal rabbit uterine fluid protein at day 6 p.c. The effect

of estradiol and progesterone on the secretion of uteroglobin

in day 6 p.c. uterine fluid was studied. It was found that

treatment with estradiol for 3 days (100, 100 and 50 pg) plus

5 mg progesterone per day for 5 days resulted in a doubling

of the relative percentage of uteroglobin present in the

uterus of pregnant rabbits on day 6 p.c. However, when exces-

sive amounts of estrogen were administered, there was a reduc-

tion in the amount of uteroglobin produced. This suggested










that uteroglobin synthesis and/or secretion was controlled

primarily by progesterone.

The hormonal control of blastokinin (uteroglobin) was

further investigated by Urzua et al. (1970) and Arthur and

Daniel (1972). They found that blastokinin was present only

in uteri of ovariectomized rabbits receiving either exogenous

progesterone or a combination of progesterone and estradiol.

This clearly indicated that the synthesis and/or secretion of

blastokinin was induced by progesterone. When blastocysts

were transferred into progesterone-treated castrate rabbits,

the pattern of blastocyst growth and differentiation up to

the time of implantation was similar to that observed in

normal pregnancy.

Urzua et al. (1970) observed that the greatest binding

of progesterone-7- 31 to rabbit uterine fluid protein was on

day 5 p.c. when there was also a peak concentration of blasto-

kinin. Sephadex G-200 gel filtration showed that the labeled

progesterone and blastokinin were eluted together. Arthur

et al. (1972) confirmed that blastokinin binds progesterone

and also estradiol, but with less affinity. The binding of

progesterone to blastokinin was found to be inhibited by the

addition of estradiol.

Goswami and Feigelson (1974) identified and character-

ized by means of electrophoretic mobility on polyacrylamide

gels a rabbit uterine specific protein which had a character-

istic cone-shaped configuration. Their "cone" protein

resembled blastokinin (utcrglobin) with respect to










electrophoretic mobility and molecular weight. However, con-

trary to the data of Urzua et al. (1970) and Arthur et al.

(1972) which demonstrated 311-progesterone binding by blasto-

kinin, they were unable to consistently demonstrate 3H-proges-

terone binding by their "cone" protein.

Hamana and Hafez (1970) examined the blastokinin content

of blastocoelic fluid and found that total protein content of

blastocoelic fluid increased sharply between days 7.5 to 8.5

p.c. The blastokinin fraction in blastocoelic fluid increased

with blastocyst age to a maximum at days 6.5 to 7.0 p.c. and

then diminished and finally disappeared completely by day

8 p.c. This suggested that its physiological function was

restricted to the period in embryonic development between

shedding of the zona pellucida and implantation. They also

suggested that the trophoblast cells absorb the protein from

the uterine fluid, since day 8 of gestation also marks the

beginning of hemotrophic nutrition of the rabbit blastocyst.

El-Banna and Daniel (1972a) incubated day 5 rabbit

blastocysts in culture medium supplemented with all combina-

tions of progesterone, day 5 pregnant rabbit uterine fluid

protein or rabbit serum protein. Blastocyst growth, uridine

uptake and amino acid uptake were significantly increased when

media was supplemented with a combination of progesterone and

macromolecular components of uterine fluids. They speculated

that transport of progesterone by macromolecules was essential

for normal early embryonic development. El-Banna and Daniel

(1972b) later studied the effects of individual rabbit uterine











fluid fractions on embryonic growth in vitro. They found

that blastokinin was consistently the most effective promoter

of blastocyst growth and incorporation of uridine and amino

acids.

Murray et al. (1972a) demonstrated the quantitative and

qualitative variation in protein constituents of swine uterine

fluids during the estrous cycle. Sephadex G-200 gel filtra-

tion revealed five protein fractions; Fractions I, II and III

which did not vary with the estrous cycle and Fractions IV

and V which were present only during the luteal phase of the

estrous cycle. The estimated molecular weights for these

five fractions were greater than 200,000, 200,000, 90,000,

45,000 and 20,000, respectively. Fraction V first appeared

on day 9 of the estrous cycle and continued through day 16.

This fraction constituted greater than 20 percent of the total

recoverable protein on days 12 through 16 and reached its peak

concentration on day 15. Polyacrylamide gel disc electropho-

resis showed that Fraction V contained at least six acidic

proteins at pll 8.0. Fraction IV, composed primarily of a

lavender colored basic protein, was present on days 12 through

16 of the estrous cycle. Murray et al. (1972) observed that

appearance of these low molecular weight uterine specific

proteins was coincidental with the rapid elongation of the

trophoblast of the pig blastocyst, which begins on day 11 of

pregnancy with rapid elongation of the blastocyst and differ-

entiation of the germ layers of the embryonic disc into

specialized t .sue. Murray (1971) further suggested that the










lavender uterine specific protein (Fraction IV) may have some

regulatory role as a repressor or inducer of genetic expres-

sion since it is basic and could theoretically interact with

nucleic acids. If so, it may play a key role in regulating

trophoblast growth and early embryonic development.

Knight et al. (1973b) undertook a study to determine the

key hormones) responsible for the quantitative and qualita-

tive changes in the protein milieu of the uterine protein

secretions in gilts during the estrous cycle. Twelve cycling

gilts were ovariectomized on day 4 of the estrous cycle and

randomly assigned to receive either progesterone (P, 2.2

mg/kg), estradiol (E, 1.1 pg/kg), progesterone and estradiol

(PE) or corn oil (C, 4 ml) daily. Injections were begun on

day 4 and continued until uterine protein secretions were

obtained by flushing the uterus on day 15 after onset of

estrus. Total uterine protein recovered was 13.7, 14.1,

77.5 and 167.3 mg for the C, E, P and PE treated gilts, respec-

tively. Highly significant (P<.01) differences in the quan-

tity of uterine protein recovered were found between the corn

oil and the progesterone treated, and the corn oil and the PE

treated gilts; between the estradiol and the progesterone

treated, and the estradiol and PE treated gilts; and between

the progesterone and PE treated gilts. However, differences

between CO and E treated gilts were not significant (P>.10).

Sephadex G-200 gel filtration protein profiles for progester-

one and PE treated gilts were similar to those for intact,

untreated, day 15 control gilts; however, protein profiles










from corn oil and estradiol treated gilts revealed the pres-

ence of only Fractions I, II and III (Murray et al., 1972a).

Fractions IV and V were present only in gilts receiving

progesterone. Polyacrylamide gel disc electrophoresis con-

firmed the Sephadex G-200 findings and further demonstrated

that uterine protein Fractions IV and V, and a component of

Fraction II, were progesterone induced. These data clearly

demonstrated that progesterone was the primary hormone respon-

sible for quantitative and qualitative changes in porcine

uterine specific protein secretions and that a synergistic

relationship between progesterone and estrogen was responsible

for maximum porcine uterine protein secretion.

Knight et al. (1973b) also examined the effect of length

of progesterone treatment on quantitative and qualitative

aspects of porcine uterine protein secretions and compared

the secretary pattern of progesterone induced uterine protein

secretions with that for untreated, intact gilts, first

reported by Murray et al. (1972a). Thirteen gilts were ran-

domly assigned to be ovariectomized on day 4 of the estrous

cycle and treated daily with progesterone (2.2 mg/kg) until

uterine flushings were obtained either on day 7, 9, 11, 13,

15, 17 or 19. The quantitative pattern of progesterone

induced uterine protein secretions was found to be quite

similar to that for intact, untreated gilts up to day 15 of

the estrous cycle. However, the quantity of uterine protein

secretions continued to increase on days 17 and 19 in the

progesterone treated group, while it decreased sharply after










day 15 (corresponding with expected time of CL regression,

Masuda et al., 1967) and reduced progesterone production

(Gomes et al., 1965; Schomberg et al., 1966; Masuda et al.,

1967; and Tillson et al., 1970) in the intact, untreated

gilts (Murray et al., 1972a). Sephadex G-200 gel filtration

also showed qualitative aspects of uterine protein secretions

to be similar to that for the intact, untreated gilt until

day 15 of the estrous cycle. Polyacrylamide gel disc electro-

phoresis revealed at least four of the six protein components

of Fraction V, Fraction IV, and one component of Fraction II

to be progesterone induced. Although precise roles of the

uterine specific proteins were not defined, it was suggested

that they may play a role in embryonic growth and differenti-

ation since their occurrence was coincidental with the period

of rapid elongation of the trophoblast of the pig blastocyst,

which begins after day 11 of pregnancy (Green and Winters,

1946).

In a subsequent study, Knight et al. (1974b) examined

the effects of varying amounts and/or ratios of progesterone

and/or estradiol on quantitative changes in porcine uterine

protein secretions in ovariectomized and sham-operated gilts.

In general, it was found that the quantity of total recover-

able uterine protein increased as the quantity of exogenous

progesterone administered increased. This increase was more

apparent and more consistent in ovariectomized gilts than it

was in sham-operated, intact gilts. This apparently was due

to the reduction or elimination of endogenous progesterone










and estrogen and thus the elimination of their possible influ-

ence on quantity of recoverable uterine protein. Highly sig-

nificant (P<.01) positive correlations were found between

total quantity of exogenous progesterone administered and the

total quantity of uterine protein recovered in ovariectomized

gilts (r=.72). Nonsignificant negative correlations were

found between estradiol dosage and the quantity of recover-

able uterine protein when the level of estradiol administra-

tion was increased. This suggested that estrogen may inhibit

and/or delay porcine uterine protein secretions when estrogen

levels rise above the level necessary for a synergistic rela-

tionship with progesterone. Support for this hypothesis was

suggested by data of Beier et al. (1972). They reported that

when 200 and 220 pg of estradiol were administered to rabbits

on the day of coitus and day 1 p.c., respectively, there was

a 2- to 5-day delay in blastokinin secretion. As a result,

the day 8 uterus of estradiol treated rabbits would support

day 4 blastocysts, but the day 4 estradiol treated uterus

would not support day 4 blastocysts.

The possible involvement of uterine specific proteins

as enzymes in development of the concepts is poorly defined

and not clearly understood. Bredeck and Mayer (1955) examined

the uterine acid and alkaline phosphatase concentrations in

rats and their relationship to number and weight of embryos.

They found that the uterine acid phosphatase concentration

was greater than the uterine alkaline phosphatase activity in

both pregnant and nonpregnant rats. An increase in uterine










weight was found to be paralleled by an increase in acid

phosphatase concentration and by a decrease in alkaline phos-

phatase concentration. There was a highly significant

(P<.01) positive correlation (r=.744) between weight of uter-

ine contents and uterine acid phosphatase content. There

were also highly significant (P<.01) positive correlations

between acid phosphatase concentration and number of implan-

tations (r=.616) and number of viable embryos (r=.635).

Wislocki and Wimsatt (1947) reported that the chorionic

epithelium absorbs material present in the uterine lumen,

including phosphatase. They suggested that phosphatase was

passed through Reichert's membrane into the yolk sac where it

was absorbed by the cells lining the visceral wall. Phospha-

tase was reported to appear gradually and then increase

steadily in amount as the placenta aged. The phosphatase

enzyme was localized chiefly in the trophoblastic elements

of the endotheliochorial trabeculae. Recent data (Bazer

et al., unpublished data) suggest that the acid phosphatase

specific activity of the uterine specific porcine purple pro-

tein (Fraction IV) isolated from the allantoic fluid of preg-

nant gilts increased steadily from day 20 until day 60 of

gestation and then decreased toward term.

Lutwak-Mann (1955) investigated the occurrence, distri-

bution and hormonal dependence of a specific endometrial

enzyme, carbonic anhydrase, in the rabbit genital tract and

blastocyst. More recent data substantiate her conclusion

that ovarian steroids reguiite oviducal and uterine enzymes.










Leucine aminopeptidase has been demonstrated to be controlled

primarily by progesterone. IIistochemical analysis has shown

maximum activity in the cavum epithelium at day 4 and 5 p.c.

in the rabbit uterus (Beier, 1970; Denker, 1971). Bazer et

al. (unpublished data) found leucine aminopeptidase activity

to be associated with porcine uterine protein Fraction II.

Kirchner et al. (1971) showed that 3-glycoprotein was a

uterine protease with maximum activity at day 7 p.c. in the

rabbit. Denker (1972) demonstrated proteolytic activity in

the rabbit trophoblast by a substrate film technique. Beier

(1974) observed that both proteases developed peak activity

during attachment and invasion of the trophoblast, and that

they seemed to be involved in this process. Regulation of

protease activity within the rabbit uterus has been reported

to be controlled by uterine protease inhibitors, the activity

of which has been shown to be stimulated by ovarian hormones

(Beier, 1970; Denker, 1972). The proteolytic enzymes cathep-

sin A, B, C and 1) and lysozyme have been found in pig uterine

fluid (Bazer et al., unpublished data).



Porcine Conceptus Development


Assheton (1906) demonstrated continued growth of uterine

glands of the pig up to 25 days of pregnancy and suggested

that the uterine glands may be of importance for nourishment

of embryos throughout gestation. Wislocki and Key (1921)

found that mitochondria were numerous in the epithelial cells










of the chorionic ectoderm and the uterine mucosa which form

the bridges between maternal and fetal tissue. Mitochondria

were also found to be abundant in the epithelium of the uter-

ine glands from which the uterine milk (Bonnet, 1882; Grosser,

1927) was believed to be liberated. This finding suggested

that an important metabolic process was carried on at this

site. Wislocki and Dempsey (1946) studied cells of the uter-

ine glands and chorion of pregnant sows. Their more signifi-

cant histochemical findings were that there was a large number

of mitochondria and large amounts of acid phosphatase, iron

and lipoidal reactions associated with these cells. It was

strongly suggested that these materials were absorbed by the

fetal chorio-allantoic membrane.

The areolae is the structure by which the embryo absorbs

secretions of the maternal uterine glands. This structure

was first described by Hunter (1781), who called it a"circular

white spot." lie observed that due to their thickness, these

spots were more opaque than the surrounding membrane. Von

Baer (1828) made a detailed study of these structures and

described them as being similar to the intercotyledonary

areas of the ruminant placenta. The chorionic depressions

were found to be opposite the depressions on the uterine wall

and each was found to terminate in a uterine gland. From

this structural relationship it was concluded that the secre-

tion of the uterine glands was absorbed by the depression on

the chorion. Eschricht (1837) was the first to term these

structures areolaee." He described the vascularization of










the areolae and clearly pointed out that the areolae lie in

direct apposition to the mouth of a uterine gland. Turner

(1875) studied the vascular pattern of both the maternal and

fetal components of areolae and he also concluded that the

areolae was the center of absorption of materials elaborated

by the uterine gland.

Heuser (1927) reported that areolae appear on the chori-

onic surface soon after the pig embryo attains a crown-rump

length of 12 mm. lie found the areolae to be small circular

spots in the early stages of embryonic development which

gradually became thicker and more conspicuous as gestation

progressed.

Goldstein (1926) stated that areolae arise as invagina-

tions of the chorionic surface, opposite the mouths of the

corresponding uterine glands, and became large saccular struc-

tures. Their function was observed to be the assimilation of

uterine milk which was poured into their cavities by the

uterine glands and, in that manner, provide nourishment for

the fetus.

Brambel (1933) reported that the first appearance of the

areolae was on the chorion of the 8 mm embryo. The areolae

were reported to arise as minute circular discs of cylindrical

cells opposite the mouths of uterine glands. lie suggested that

areolae arise as the result of a stimulus from uterine gland

secretions. He made a detailed study of the distribution of

areolae throughout gestation and found that the total number

of areolae increased as gestation progressed, but not with










regularity of increment. The total number of areolae per

square centimeter of allantochorionic surface increased very

rapidly from the 8 mm fetal crown-rump length stage to the

30 mm stage, reached a plateau by the 150 mm stage and then

declined to term. The distribution of the areolae on differ-

ent zones of the chorion was also studied. It was found that

in the equatorial (interior) zones, the areolae increased in

number very rapidly, especially up to the 30 mm stage. The

areolae were found to make their first appearance in the cen-

tral regions of the allantochorion, and for a time no areolae

were found on the polar sections. As gestation progressed,

areolae in the interior zones of the allantochorion became

more structurally complex than those on the polar sections.

The total areolae area increased as.gestation progressed, due

to an increase in total number of areolae and an increase in

size of each areolae. Brambel (1933) supported the suggestion

of previous workers that areolae serve as sites of absorption

and transport of secretions of the uterine glands to the

embryo. lie also suggested that the inter-areolar area was

the site of exchange of respiratory gases and that the inter-

areolar area became increasingly important functionally as

gestation progressed. Recently, Chen et al. (1975) utilized

immunofluorescent antibody techniques to confirm that areolae

are the site of absorption and transport of uterine gland

secretions. They have demonstrated that the purple acid

phosphatase in pigs is secreted by the uterine endometrial











glands, transported across the chorio-allantoic membranes via

the areolae and sequestered in the allantoic fluid.

Wislocki (1935) studied the changes in allantoic and

amniotic fluid volume during the course of gestation in the

sow. le reported a very dramatic early increase in allantoic

fluid volume between the 10 mm crown-rump length stage

(approximately day 20 of gestation) and the 25 mm stage

(approximately day 30 of gestation). The increase in volume

was from less than 10 ml to greater than 200 ml. This early

rapid increase in allantoic fluid volume was followed by a

subsequent rapid decrease to about the 80 mm stage (approxi-

mately day 45 of gestation). There was then a second rapid

increase in allantoic fluid volume to about the 120 mm stage

(approximately day 60 of gestation)' and a subsequent decrease

towards term. Based upon these rather dramatic allantoic

fluid volume changes, Wislocki (1935) did not agree with the

theory that allantoic fluid was primarily a result of excre-

tory products of the mesonephros. In studying amniotic fluid

in sows, he found that the volume reached a maximum at about

the 190 mm stage (approximately day 70 of gestation) and then

underwent a slight, although variable, decrease towards term.

McCance and Widdowson (1953) suggested that allantoic

fluid was secreted by the placenta. The composition of the

fetal fluids at 20, 46 and 65 days of gestation were analyzed

by McCance and Dickerson (1957). Based upon differences

which they observed between the allantoic and amniotic fluid

composition compared with that of maternal and fetal serum,










they suggested that the fluids were formed by the process of

secretion rather than dialysis.

Hammond (1914, 1921) first reported that the size of the

fetal membranes was directly proportional to the size of the

fetus. Unfortunately, he erroneously concluded that the

vitality of the placenta depended upon that of the fetus,

rather than vice versa, lie also found that average weight

of the embryo decreased as size of the litter increased. The

number and proportion of dead fetuses in relation to the total

number of fetuses found in the horn was also recorded. lHe

noted a trend towards a larger proportion of dead fetuses in

the most crowded horns at stages of gestation beyond day 30.

Warwick (1928) studied the pattern of prenatal growth of

swine and reported growth curves for length and weight of the

fetus and weight of the placenta. A direct relationship

between placental and fetal size was found. Fetal crown-rump

length and fetal weight were found to be correlated with pla-

cental weight at all stages of gestation. Waldorf et al.

(1957) and Pomeroy (1960) later reported similar significant

correlations. Total distance between fetuses and placental

weight were also found to be positively correlated, suggest-

ing that crowding had some effect in retarding development

of the placenta. It was also found by Warwick (1928), and

later substantiated by Pomeroy (1960), that the placenta

attained its maximum size by approximately day 60 of gesta-

tion, and grew very little, if any, thereafter. This sug-

gested that fetal growth in the latter stages of pregnancy










was largely dependent upon the placental development which

had occurred during the first 60 days of pregnancy. The

growth curves showed that fetal length increased at a fairly

uniform rate throughout the gestation period, while fetal

weight increased much more rapidly during the last 20 days

of gestation than it had earlier. Weight of the fetus first

reached that of the fetal membranes between 60 and 70 days of

gestation. It was found that both a large number of fetuses

per horn and a small average space per fetus was accompanied

by a marked increase in number of degenerate fetuses, particu-

larly in the latter stages of gestation. This implied that

crowding, probably due to its restriction on placental devel-

opment, was related to increased embryonic death.

Barcroft (1944) concluded that.size of the fetus was

limited largely by size of the placenta, and more particularly

by the size of its vascular bed. Pomeroy (1960) found that

the effect of litter size on variability of fetal weight in-

creased after about 60 to 70 days of gestation which corre-

sponded to the stage when size of the placenta had reached

its maximum. He concluded that since the fetus was dependent

upon the placenta for its nutrition, fetal growth was likely

to be conditioned by the ultimate size of the placenta, which

in turn was affected by the number of fetuses present prior

to 60 days of gestation. Ile also found that each additional

fetus resulted in a reduction in average fetal weight of

approximately 7.7 grams (g) when the fetal weights were

determined after day 70 of gestation.










Waldorf et al. (1957) found that as the number of fetuses

per uterine horn increased there was an insignificant decrease

in average fetal weight at day 105 of gestation. They sug-

gested that this relationship was directly related to the

amount of space available for placental development.

In a study of day 65 guinea pig fetuses, Eckstein and

McKeown (1955) found that as litter size increased, mean

fetal weight and length decreased while total litter weight

increased. Also, mean fetal weight and length decreased as

the number of fetuses in the same horn increased. In a sub-

sequent study, Eckstein et al. (1955) reported that placental

weight was inversely related to both the number of fetuses in

the same uterine horn and the number of fetuses in the oppo-

site uterine horn. In litters with unequal numbers of fetuses

in the two uterine horns, placental weight was greater on the

side with fewer fetuses.

Perry and Rowell (1969) demonstrated that when the number

of fetuses present in a uterine horn exceeded five, those at

the ends of the uterine horn tended to have an increasing

fetal weight advantage over those in the middle (up to 5 to

10 percent heavier) and the fetuses at the ovarian end tended

to have an increasing weight advantage over those at the

cervical end (up to 10 to 15 percent heavier). This was in

general agreement with an earlier report by Waldorf et al.

(1957) that the fetuses and membranes at the extremes of the

uterine horn were larger than those toward the middle.










The effect of the amount of placental attachment area on

fetal growth and survival was examined by Rathnasabapathy

et al. (1956). Their examination of 195 fetuses from gilts

slaughtered on day 55 of gestation showed a highly significant

(P<.01) positive correlation (r=.552) between fetal weight

and the amount of uterine space occupied by the concepts unit.

Based upon the relationship which they found between fetal

weight and uterine space, they suggested that overcrowding

was an important cause of fetal death. They found a curvi-

linear relationship between fetal weight and uterine space.

When uterine space was 50 to 100 mm, average weight of the

fetus was 41.88 g. An addition of 50 mm uterine space

resulted in a weight increase of 18.74 g. Every subsequent

addition of one unit space up to 200 mm was then associated

with a 10 g increase in fetal weight. Further additions in

length up to 400 mm brought less than a 10 g weight increase

in the fetus. Beyond 400 mm, additional uterine space

resulted in no further increase in fetal weight. It was esti-

mated that an optimum uterine space of 350 to 450 mm was

necessary for every fetus for maximum growth at the stage of

pregnancy studied (55 days). Inadequate space was believed

to result in fetal atrophy. A positive and highly signifi-

cant (P<.01) correlation (r=.406) was observed between total

uterine length and fetal survival. It should be pointed out,

however, that it was impossible to determine if the uterus

was inherently long or long due to the stimulatory influence

of a large number of fetuses, although the latter was probably










the case. Dhindsa et al. (1967) and Rigby (1968) reported

large individual variations in uterine horn length in non-

pregnant gilts.

Wrathall (1971), in a review of embryonic mortality in

pigs, concluded that embryos which obtain small areas of

placental attachment in the early stages of gestation may

have no further opportunity to increase their relative pla-

cental size later in gestation and thus remain at a disadvan-

tage throughout gestation. lie suggested that competition for

uterine attachment area during the second and third weeks of

gestation had far-reaching and important permanent influences

on fetal development, litter size and ultimately even on post-

natal survival. Since the placenta of the pig grows very

little after approximately day 60 of gestation and fetal

growth occurs most rapidly in the later stages of gestation

(Warwick, 1928; Pomeroy, 1960) and since fetal growth is pri-

marily dependent upon placental growth (Warwick, 1928; Bar-

croft, 1944; Rathnasabapathy et al., 1956; Waldorf et al.,

1957; and Pomeroy, 1960), the significance of developing ade-

quate placental mass in early gestation is obvious.

Knight et al. (1974a) studied the effect of a progester-

one induced increase in uterine secretary activity on develop-

ment of the porcine concepts. Thirteen gilts were bred at

estrus and then either bilaterally ovariectomized or sham-

operated on day 4 after onset of estrus. Treatments imposed

were: (1) sham-operated, corn oil (SO-CO); (2) sham-operated,

3.3 mg progesterone (P) and 0.55 pg estradiol (E)/kg/day










(SO-HP); (3) bilaterally ovariectomized, 1.1 mg P and 0.55 pg

E/kg/day (OVX-LP) or (4) bilaterally ovariectomized, 3.3 mg P

and 0.55 pg E/kg/day (OVX-IIP). These treatment levels in

contemporary gilts had previously been demonstrated (Knight

et al., 1974b) to result in a highly significant (P<.01) in-

crease in quantity of recoverable uterine protein from non-

pregnant SO-IHP and OVX-IIP treated gilts as compared to the

SO-CO and OVX-LP treated gilts. On the basis of these data,

it was assumed that secretion of uterine proteins would be

increased in a similar manner for pregnant gilts and allow

the comparison of high and low levels of uterine protein

secretion on concepts development. Treatment was maintained

from day 4 to day 40 of gestation when all gilts were hyster-

ectomized. Results indicated that placental length was sig-

nificantly (P<.05) increased by approximately 7 cm in the

SO-HIP and OVX-IIP treated gilts compared with the SO-CO and

OVX-LP treated gilts. Allantoic fluid volume was also sig-

nificantly (P<.05) greater in the SO-lIP vs. SO-CO and in the

OVX-IIP vs. OVX-LP groups. Placental weights were significantly

(P<.05) greater in the sham-operated compared to the bilater-

ally ovariectomized females and the SO-lHP treated gilts had

significantly (P<.05) heavier placentae than those from gilts

in the other three treatment groups. Since an increase in

placental length precedes the increase in placental weight

in normal development of the porcine placentae (Warwick, 1928;

Pomeroy, 1960), it was felt that this experiment may have been

terminated too early to fully evaluate the effect of these










treatments on placental weight. Collectively, these data

suggested that increased secretary activity, which resulted

from the high progesterone level, enhanced placental develop-

ment. The establishment of maximum placental surface area

was felt to be of critical importance with respect to fetal

growth and survival as pregnancy progressed towards term.

Chen (1973) examined the influence of sheep antiserum

against porcine lavender uterine protein (Fraction IV, Murray

et al., 1972a) on embryonic and placental development in early

and mid-pregnancy. A series of experiments were conducted

employing passive immunization of gilts systemically and by

injecting the antisera directly into the allantoic fluid of

pregnant gilts. Intravenous injection of sheep anti-lavender

uterine protein antisera into pregnant gilts in early gesta-

tion (days 7, 9, 11, 13 and 15) significantly (P<.01) inhibited

placental growth as indicated by a reduction in placental

length at day 30 of gestation (49.43 cm in treated vs. 58.99

cm in control gilts). Embryonic development was not affected.

Attempts to study the effects of injecting the antisera

directly into the allantoic fluid were unsuccessful since the

experimental method led to abortion, i.e., injections of

saline also caused abortion. A passive intravenous immuniza-

tion with the sheep anti-lavender protein antisera during

later stages of pregnancy (days 34, 36, 38, 40 and 42)

resulted in a significant (P<.01) reduction in placental

weight (95.94 g for treated vs. 126.67 g for control gilts)

and placental length (58.92 cm for treated vs. 68.76 cm for










control gilts) at 50 days of gestation. Again, development

of the embryo was not affected. These data suggested that

porcine lavender uterine protein may influence the developing

embryo by stimulating or enhancing trophoblast growth during

the period of rapid growth of the blastocyst and development

of the chorio-allantoic membranes during the second month of

pregnancy.



Progesterone and Estrogen Levels in Pigs


During the Estrous Cycle

Although the absolute levels of plasma steroids at a

given stage of the estrous cycle are highly variable between

investigators and laboratories, depending upon the type,

specificity and sensitivity of the assay procedure, as well

as the accuracy and precision of the investigator, general

patterns of change in plasma progesterone levels reported in

the literature for swine are similar. At estrus the proges-

terone level is at a basal level (<1 ng/ml plasma). There is

then a rapid increase between days 2 and 6 of the estrous

cycle (corresponding with CL formation and development) and a

continued increase to a maximum plasma progesterone concen-

tration at days 12 to 14 of the estrous cycle. A very pre-

cipitous decrease in plasma progesterone occurs after day 15

in association with rapid CL regression, and remains at a

basal level until diestrous of the next cycle (Gomes et al.,

1965; Masuda et al., 1967; Tillson and Erb, 1967; Stabenfeldt










et al., 1969; Edqvist and Lamm, 1971; Guthrie et al., 1972;

Ilenricks et al., 1972; and Shearer et al., 1972).

Estimates of plasma estrogen concentrations during the

estrous cycle of swine are also variable between investigators.

In addition to the reasons for variation pointed out in the

discussion of plasma progesterone concentration, the estrogen

levels also vary depending upon whether total estrogens,

estradiol or estrone were measured. However, the general

pattern of change in plasma estrogen concentration during the

porcine estrous cycle found by most investigators is similar

to the data of Henricks et al. (1972). They measured total

estrogens by radioimmunoassay techniques. Plasma estrogen

concentrations in pigs ranged from 10 to 30 pg/ml throughout

much of the estrous cycle except for the day prior to estrus

when there was an increase in concentration to 60 to 70 pg/ml.

The level then declined and remained at basal levels until

day 14 or 15 of the subsequent estrous cycle when a slow

increase began and continued until 2 days prior to estrus.

Generally, the plasma estrogen concentration began to rise

coincidentally with the disappearance of plasma progesterone.

At two days prior to estrus, the level increased sharply.

On the day of estrus the level declined in some gilts, but

this decline was quite variable. The estrogen concentration

reached a maximum level about 48 hours prior to the time the

maximum plasma luteinizing hormone (LH) concentration occurred

in association with onset of estrus. This general pattern

agrees with the findings of Lunaas (1962), Raeside (1963a),










Bowerman et al. (1964), Guthrie et al. (1972) and Shearer

et al. (1972).


During Gestation

The general pattern of change in peripheral plasma pro-

gesterone concentration in the pregnant pig is as follows:

progesterone concentrations from the day of onset of estrus

until day 12 to 14 of pregnancy are essentially identical to

those reported for the nonpregnant pig, i.e., a rapid increase

between days 2 and 6 and a continued increase to a maximum

level at days 12 to 14. However, after this early peak in

the pregnant gilt there is a steady decline in progesterone

until approximately day 25 of gestation. Reports of the

magnitude of this decrease range from 30 to 50 percent

(Masuda et al., 1967; Guthrie et al., 1972; Guthrie et al.,

1974; and Robertson and King, 1974). From approximately day

25 until the approach of parturition, the plasma concentra-

tion of progesterone remains fairly constant (Robertson and

King, 1974). All studies of progesterone concentrations in

the late stages of gestation have indicated a decline as par-

turition approached, but the reported time at which this

decrease began varied between investigators. Robertson and

King (1974), who were the only investigators that made a

systematic study of the changes in progesterone concentration

throughout all of gestation, reported the progesterone con-

centration to gradually decline over the last 15 days of ges-

tation with a further sharp decline occurring immediately










prior to parturition. The progesterone concentration de-

creased to less than 0.5 ng/ml within 24 hours after farrow-

ing. Data from Molokwu and Wagner (1973) indicated that the

progesterone level began to decline approximately 4 or 5 days

before farrowing and then decreased very abruptly during the

last 48 hours prior to farrowing. In most animals in their

study the progesterone levels were below 1 ng/ml in peripheral

plasma at parturition. Killian et al. (1973) reported that

progesterone levels were quite constant during the last 3

weeks of gestation but declined rapidly from 10 ng/ml at 3

days prepartum to less than 1 ng/ml by day 1 postpartum.

Ash et al. (1973) reported a gradual decline in progesterone

concentration over the last 7 days of gestation and a sharp

decrease within 48 hours before parturition.

Estrogens in the pregnant pig have been shown to undergo

a transient rise between days 20 and 30 of gestation. Follow-

ing this initial rise, estrogen levels then decreased and

remained relatively stable until a second rise began at

approximately day 70 of gestation. This increase continued

at a rapid rate until parturition (Velle, 1960; Lunaas, 1962;

Raeside, 1963b; Bowerman et al., 1964; Guthrie et al., 1972;

Ash et al., 1973; Lunaas et al., 1973; Molokwu and Wagner,

1973; Guthrie et al., 1974; and Robertson and King, 1974).

Velle (1960) first reported that urinary estrone levels

increased between 24 and 32 days of gestation and suggested

that quantitative determination of this increase was a










reliable method of pregnancy diagnosis in swine. Lunaas (1962)

substantiated this early urinary estrone increase.

Raeside (1963) and Bowerman et al. (1964) examined uri-

nary estrogen levels throughout the gestation period. Both

reported that estrone was the principal component of the

estrogens in pregnant gilts and sows. They also confirmed

the early peak of estrogen at approximately 30 days of gesta-

tion and reported that urinary estrogens decrease following

this initial peak, remain relatively constant until day 70

and then undergo another increase that continues until term.

Ash et al. (1973) reported a rapid increase in peripheral

plasma estrogens during the last week of gestation. They

found that the peripheral estrogen concentration was greater

in multiparous animals and in sows carrying a large number of

fetuses. The plasma estrogens were found to consist mainly

of estrone and small quantities of estradiol-170.

Lunaas et al. (1973) reported the same early increase in

conjugated estrone levels in uterine venous blood which had

been previously reported for urinary estrogens. They sug-

gested that the ovarian secretion of estrogens during preg-

nancy was negligible. They observed that the estrogen pro-

duction which apparently took-place in the concepts during

pregnancy coincided with a rapid expansion of the allantoic

membranes and that the allantoic fluid also contained high

levels of conjugated estrone.

Molokwu and Wagner (1973) studied the changes in estro-

gen concentration in the pcuI pheral plasma of the sow










approaching parturition and found a substantial increase the

last week before parturition. Estrone levels increased from

1,200 pg/ml to a peak of 2,368 pg/ml by 2 days prepartum.

This level was essentially maintained through the time of

farrowing and then declined rapidly to 200 pg/ml at 24 hours

postpartum and by one week postpartum was approximately 6 to

7 pg/ml. Estradiol showed similar relative changes although

the peak level for estradiol was only 75 to 80 pg/ml just

prior to farrowing. Again the levels declined very rapidly

after farrowing and were at a basal level of 4 to 5 pg/ml by

one week postpartum. The estrogen:progesterone ratio showed

an increase which became increasingly rapid as parturition

approached.

Robertson and King (1974) reported unconjugated estrone

and estradiol-178 to rise sharply between day 80 of gestation

and parturition. Peak levels of 2.5 ng/ml estrone and 400

pg/ml estradiol were found. Plasma concentrations of estrone

sulfate were reported to increase from 60 pg/ml at day 16 to

greater than 3 ng/ml between days 23 and 30 of pregnancy.

The plasma concentration of estrone sulfate then declined

sharply to a low value of 35 pg/ml around day 46 of gestation

before slowly rising again to attain a peak of 3.0 ng/ml on

the day before parturition. Concurrent determination of

unconjugated estrone indicated that only 15 pg/ml were present

at the time (days 23 to 30) when the concentration of estrone

sulfate was 3 ng/ml. They suggested that the concepts was

the source of estrone sulfate.










Perry et al. (1973) demonstrated that the pig blastocyst

has the capability of steroid hormone production. They found

no significant incorporation of tritium-labelled androstenedi-

one into estrogens by the day 10 blastocyst, but by days 14,

15 and 16 the conversion of labelled dehydroepiandrosterone

(1)IIA) or androstenedione to estrone and estradiol-173 by

blastocysts was appreciable. The rate of incorporation of

)IIA into estrogen was about twice that for androstenedione.

The overall ratio of estrone to estradiol-177 was about 7:1

for both precursors. A low rate of conversion of labelled

progesterone to estrogens was also demonstrated; however,

there was no detectable incorporation of labelled cortisol

or labelled pregnenolone into estrogens. These data provided

biochemical evidence for the presence, within the blastocyst

tissue, or aromatase, 17-20 desmolase and 3-sulphatase enzyme

systems concerned with the production of estrogens from

neutral steroids, progesterone and conjugated steroids,

respectively. These data indicated that the presence of

unconjugated estrogen and progesterone in blastocyst tissue

was probably a result of their synthesis in situ and not a

result of their diffusion from the maternal circulation.

Therefore, as early as the blastocyst stage, the pig concepts

has the enzymatic capability to produce biologically important

steroids.

Dickmann and Dey (1974) have also demonstrated that the

preimplantation rat embryo possesses steroidogenic capabili-

ties. Based upon their findings that (1) transformation of











morulae to blastocyst occurred in hormone free media in vitro;

(2) morulae developed into blastocysts subsequent to their

transfer into uteri of ovariectomized rats and (3) the trans-

formation of morulae to blastocysts was blocked in pregnant

rats whose estrogcn/progesterone balance was experimentally

disturbed, they concluded that (1) transformation of morula

to blastocyst depends, at least in part, on stimulation of

steroid hormones produced by the morula and (2) the embryo

exerts a local hormone effect on the endometrium which is

critical for implantation.

Edgerton et al. (1971) examined the relationship between

litter size and the rate of excretion of six urinary metabo-

lites of estrogen and progesterone at weekly intervals from

day 19 to 110 of gestation. They found that the rate of

excretion of total estrogens increased significantly (P<.01)

as litter size increased at 26 days of pregnancy but not at

other weekly intervals. There was a significant positive

relationship between litter size at farrowing and estrone

excretion at day 26 of pregnancy. Total pregnane (sum of

four pregnane compounds: 5 -pregnan-3B-ol-20-one, 5B-pregnan-

3--ol-20-one, 5B-pregnan-3-, 20x-diol and 5B-pregnan-3-,

6--diol-20-one) excretion rate was higher for larger litters

at each weekly interval from 19 to 54 days of pregnancy

(P<.01) at 33 and 40 days) and was generally lower from 61

to 96 days. Ratios for total pregnanes to total estrogens

were lower (P<.01) at 33 days for litters of nine piglets

than litters with a greater or lesser number. At 40 days










(P<.05) and 75 days (P<.01) these ratios increased as litter

size increased. They suggested that at least during certain

periods of gestation the concepts units may metabolize

ovarian steroids to other compounds.

Monk and Erb (1974) examined the effects of uterine

crowding and altered numbers of CL to embryo ratios on CL

progesterone, excretion of steroid metabolites in urine and

embryo survival at 27 to 35 days gestation. They found that

fetal-placental units above three have no positive effect on

CL function or rate of progesterone metabolism as measured

at 26 to 27 days of pregnancy. Rate of excretion of estrone

in the urine was more dependent on the number of fetal-

placental units than the rate of excretion of pregnanes.

These results suggested that the ovaries are not a major

source of estrogen at this period of pregnancy.

A survey of the literature has demonstrated that litter

size can be increased at 25 to 40 days of gestation by hor-

monal superovulation with pregnant mare serum gonadatrophin

(PMSG) or embryo superinduction. However, increased fetal

mortality occurred during the later stages of gestation so

that litter size at term was not increased. Similarly,

reproductive performance in unilaterally hysterectomized-

ovariectomized (UHOX) gilts was similar to that for control

gilts at 25 to 30 days of gestation, but increased embryonic

mortality occurred during the later stages of gestation in

UIIOX gilts and thus litter size was reduced.










It has been shown that intra-uterine crowding during the

early stages of gestation inhibits placental development.

This placental insufficiency may in turn limit fetal growth

in the latter stages of gestation and, in extreme cases of

placental insufficiency, result in fetal death. Placental

size and fetal size have been shown to be highly correlated.

Uterine protein secretions, which have been shown to be

under hormonal control, have been demonstrated to be necessary

for embryonic survival. Data suggests that porcine uterine

specific proteins may be involved in placental growth and

development.

Fluctuations in steroid hormone levels have been shown

to occur during gestation. Recent data suggest that the

concepts, even as early as the blastocyst stage, possesses

the capability for steroidogenesis and thus may contribute

directly to or even control the changes which occur in the

pattern of steroid hormone secretions. Additional studies

are necessary to more fully delineate the interrelationships

of the steroid hormone changes and the changes in concepts

development.
















CHAPTER II

CONCEPTS DEVELOPMENT IN INTACT AND UNILATERALLY
IIYSTERECTOIIZJE )-OVARIECTObMIZED GILTS:
INTERRELATIONSHIPS BETWEEN HORMONAL STATUS,
PLACENTAL DE1VELOPMENT'I, FETAL FLUIDS AND FETAL GROWTH



Although there have been several examinations of various

aspects of porcine concepts development at specific stages

of gestation, limited data are available with regard to the

interrelationships and interdependence of the changes which

occur during the normal course of fetal growth. Likewise,

although the steroid hormone concentrations have been examined

early and late in gestation, only one investigation has been

reported in which those changes were characterized throughout

gestation (Robertson and King, 1974) and no attempt has been

made to relate these steroid hormone changes to changes in

concepts development. Additionally, although it has been

well documented that intra-uterine crowding results in

increased embryonic mortality (Hammond, 1921; Perry, 1954;

Rathnasabapathy et al., 1956; King and Young, 1957; Hunter,

1966; Dziuk, 1968; Fenton et al., 1968; Longenecker and Day,

1968; Pope et al., 1968; Bazer et al., 1969a,b,c; Monk and

Erb, 1974; and Webel and Dziuk, 1974), there are limited data

available to explain the causes) of this increased embryonic

mortality and how concepts development under conditions of










intra-uterine crowding differs from that under normal condi-

tions.

The objectives of the present study were (1) to charac-

terize porcine concepts (placental membranes, fetal fluids

and fetus) development during gestation with special emphasis

on the interrelationships and/or physiological significance

of these changes; (2) to determine the circulating and fetal

fluid levels of progesterone, estrone and estradiol during

gestation in IC and UHOX gilts and to relate changes in these

steroid hormone concentrations to various aspects of concepts

development; (3) to compare patterns of concepts growth and

development in intact control and unilaterally hysterectomized-

ovariectomized gilts in order to determine the effects of

intra-uterine crowding on concepts. development and (4) to

determine the time and cause of embryonic mortality following

intra-uterine crowding.



Materials and Methods


Experimental Design

A total of 88 sexually mature crossbred gilts of similar

age, weight and genetic background were randomly assigned to

one of two treatment groups: either (1) Intact Control (IC)

or (2) Unilaterally Ilysterectomized-Ovariectomized (UIIOX).

The only treatment difference imposed was that the UIIOX gilts

had one randomly selected uterine horn and its ipsilateral

ovary removed between days 7 and 11 of the estrous cycle prior










to mating. Therefore, the UIIOX gilts had only one-half the

uterine endometrial surface area available for concepts

development as compared to the IC gilts. Gilts in both

treatment groups were checked daily for estrus activity with

intact mature boars and were bred at 12 and 24 hours after

onset of estrus (the first day of estrus was designated day 0)

and randomly assigned to be hysterectomized on either day 20,

25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 of gestation. Four

gilts in each of the two treatment groups were hysterectomized

at each of the 11 stages of gestation.


Data Collection Procedures

The gilts were not fed for 24 to 48 hours prior to sur-

gery. At surgery initial anesthesia was induced with a 5 per-

cent sodium thiopental solution injected into an ear vein.

Anesthesia during surgery was maintained with methoxyflurane.

The reproductive tract was exposed by mid-ventral laparotomy

and the ovary or ovaries, oviduct(s), uterine horn(s), uterine

body and a portion of the cervix were removed.

The uterus was dissected free of the ovary or ovaries,

oviduct(s) and mesometrium and the length of each uterine

horn was measured from the tubo-uterine junction to the junc-

tion of the uterine body and the cervix, using a metric ruler.

The total length of each uterine horn was recorded in centi-

meters (cm). In the IC gilts, the length of each horn was

added together to determine total length of the uterine horns.

The number of corpora lutei (CL) on the ovary or ovaries










was counted, and the number of CL was assumed to represent

the ovulation rate and therefore the number of potential

embryos. The uterus was carefully dissected open along the

mesometrial border and each concepts was removed intact and

placed into a clean pan.

An uncontaminated 20 ml sample of allantoic fluid was

obtained by puncturing the chorio-allantois with an 18 gauge

needle attached to a 20 ml syringe. The chorio-allantois was

then cut, the allantoic fluid collected in the pan and,

finally, the allantoic fluid volume was measured directly in

a graduated cylinder. A sample of amniotic fluid, when pres-

ent, was obtained in the same manner, and the total volume

was measured in a graduated cylinder. The allantoic and

amniotic fluid samples obtained were labelled and frozen at

-20'C until analyzed for protein and hormone concentrations.

Protein concentration was determined using the method of Lowry

et al. (1951). Total protein values for each fetus were

derived by multiplying the protein concentration by the total

volume of fluid.

Each fetus was dissected free of the placental membranes

and fetal crown-rump length measured directly in millimeters

(mm) using a metric ruler. Care was taken to measure the

crown-rump length while the fetus was in a normal fetal posi-

tion. After the crown-rump length was determined, each fetus

was placed in an individual weighing pan or onto a piece of

aluminum foil, the tare weight of which had previously been

determined. The fetus was then weighed on an analytical










balance, the tare weight subtracted from the total weight and

the weight of each fetus, identified by its position in the

litter, was recorded. Embryo dry weight was determined by

drying each embryo to a constant dry weight, generally a

minimum of 72 hours, at 1000C in a drying oven. Fetal wet

weight was divided into fetal dry weight and that figure was

multiplied by 100 to determine percent dry matter. Percent

dry matter was subtracted from 100 to obtain percent moisture

in each fetus.

Placental length was determined by direct measurement

using a metric ruler. The placenta was extended its full

normal length for this measurement, but care was taken not to

stretch the placenta. Only that portion of the placenta

which was considered to be functional was included in the

measurement, i.e., the necrotic tips of the chorion were not

measured. Placentae from fetuses determined to be dead or

dying were not included in the calculations of any of the

placental parameters. Placental weight was determined by

weighing each placenta directly on an analytical balance.

The placenta was then individually identified and placed

in buffered formalin until they could be evaluated to deter-

mine placental water displacement volume, placental surface

area, number and distribution of areolae and total areolae

surface area. These placental measurements were only made

for placentae obtained between days 35 and 100 of gestation

since placentae prior to day 35 were too fragile.











Placental water displacement volume was measured by

placing each placenta into a graduated cylinder partially

filled with water and measuring the volume of water displaced

in milliliters (ml). Placental surface area was determined

by spreading each placenta out on heavy paper and then cutting

around the edge of the placenta with scissors. The surface

area of the paper was measured with a planimeter, and this

value was used to represent placental surface area.

Each placenta was then divided into four sections of

approximately equal size. Two sections represented the inte-

rior and two sections represented the outer or polar pieces

of the placenta. In each of the four sections, the total

number of areolae were counted in two different areas measur-

ing 2.54 x 7.62 cm each. In each area the diameter of ten

randomly selected areolae was measured. Counting of the

areolae and measurement of their diameter was done under a

dissecting microscope with an eye-piece micrometer. Knowing

total placental area, an estimate was then made of total

number of areaolae. Total number of areolae was multiplied

by the estimated surface area of each areolae to obtain an

estimate of total areolae surface area. These determinations

were made for both the interior and polar sections as well as

for the total placenta.

After the contents of the uterus had been removed, width

of each uterine horn was determined at six different randomly

selected points. The averi;ie uterine width was then multi-

plied by the uterine length to estimate the total uterine










endometrial surface area. Uterine surface area per fetus was

determined by dividing total uterine surface area by the

number of fetuses.

Empty uterine weight was determined by weighing the

uterus on an analytical balance after the contents of the

uterus had been removed.

Each fetus was examined and from this the number of live,

dead and total fetuses were determined. Percent fetal survi-

val was determined by dividing the number of corpora lutea

into the number of live embryos and multiplying that figure

by 100.

Immediately prior to hysterectomy, plasma samples were

taken from the radial vein in the leg, from a uterine vein

and from a uterine artery. Plasma samples were also taken

from fetal umbilical vessels after day 60 of gestation and

pooled within gilts. Total protein was determined on the

plasma samples from these four sources using the method of

Lowry et al. (1951), and radioimmunoassay techniques were

utilized to analyze all plasma samples for progestin, estrone

and estradiol concentrations. Equal aliquots of uncontami-

nated allantoic and amniotic fluid from each fetus were

pooled within gilts and the allantoic and amniotic fluid

samples were also analyzed for progestin, estrone and estra-

diol concentrations. Total progestins, estrone and estradiol

in the allantoic and amniotic fluids were estimated by multi-

plying the steroid hormone concentration by the total fluid

volume.










Hormone Assay Procedures

Plasma progestins were analyzed by the radioimmunoassay

(RIA) procedure described by Abraham et al. (1971). Valida-

tion of the accuracy and precision of the progestin assay

used in the present study was described by Chenault et al.

(1975). Approximately 3,000 dpm (counting efficiency equals

30 percent) or progesterone (preg-4-ene-3, 20 dione)-l, 2-3H

(50.3 Ci/mM) were added to a 16 ml conical centrifuge tube,

dried under air, and .5 ml of plasma was added to the tube,

heated for 2 to 3 minutes in a 45C water bath, and gently

mixed to achieve equilibrium with tracer steroid. Five

volumes (2.5 ml) of freshly distilled iso-octane was added to

each tube for the extraction of progesterone. Each sample

was vortexed individually for approximately 1 minute, frozen

at -200C to achieve separation of the phases, and the iso-

octane phase poured off into a second conical centrifuge tube.

This extraction procedure was repeated three times and the

extracts pooled. The pooled iso-octane extract was dried at

45C under air and stored at 40C until assayed. The dried

extract was redissolved in an appropriate volume of freshly

distilled iso-octane, a one-tenth aliquot was removed for

assessing percent recovery of tracer, three aliquots of vary-

ing amounts were removed for assay and an undiluted aliquot

was saved in case it was needed for reassay at a later date.

Pooled allantoic and amniotic fluid samples were assayed for

progestins in an identical manner to the plasma samples except

that larger volumes (1 to 2 ml) were extracted.










The progesterone antibody used in this study was a gift

from D. K. Mahajan of Pennsylvania State University. With

50 percent displacement of progesterone-1,2- 3I as a measure

of percent cross reactivity of the progesterone antiserum,

cross reactivities for 17 -01O progesterone, 20 a-OH progester-

one, 20 -011 progesterone, corticosterone, cortisol, andro-

stendione, dchydroepiandrosterone, testosterone and 11 desoxy-

corticosterone were 0, 14.2, 0, 0.8, 0.5, <0.1, <0.02, 0.5

and 3.0 percent, respectively. Based upon the low cross

reactivities found between the progesterone antibody used and

other progestins, it is believed that these determinations

essentially represent progesterone. However, the estimates

reported in this study will be defined as progestins, not

progesterone.

Estrone and estradiol in all plasma, allantoic and amni-

otic fluid samples were also measured by RIA. The estradiol

assay procedure and validation used in this study was described

by Chenault et al. (1975). The estrone assay procedure dif-

fered from the estradiol only in regard to the antiserum

dilution (1:30,000 vs. 1:40,000) and the time of incubation

(overnight vs. 3 hours). Approximately 3,000 dpm of estradiol-

17B (estra-1,3,5(10)-triene-3,17B-diol)-2,4,6,7-311 (106 Ci/mM)

and of estrone (3-hydroxy-estra-1,3,5(10)-triene-17-one)-2,4,

6,7-3If (106 Ci/mM) in ethanol were added to 30 ml, 18 x 150 mm

screw cap test tubes with teflon-lined caps and dried.

Depending upon the fluid pool assayed and the stage of gesta-

tion, 0.5 to 5.0 ml were addod and allowed to achieve










equilibrium with the isotope. Samples were extracted with

two volumes of diethyl either from a freshly opened can.

Extraction tubes were gently shaken for 1 to 2 minutes, fro-

zen at -200C, and the ether phase poured off into 50 ml coni-

cal centrifuge tubes. The extraction was repeated three times,

and pooled ether extracts were evaporated to dryness under

air. The tubes were then rinsed with a small volume of ben-

zene:methanol (90:10) to concentrate the extract within the

tips of the conical tube. This volume was dried under air.

Separation of estrone and estradiol was accomplished by

Sephadex LI-20 column chromatography. Sephadex LH-20 was

soaked at least 4 hours in benzene:methanol (90:10) solvent.

Chromatographic columns were prepared by placing a filter

disc in the bottom of a 2.5 ml glass tuberculin syringe.

Approximately 1 ml of solvent was added to the syringe to

moisten the filter disc. Using a Pasteur pipette, a slurry

of presoaked Sephadex LH-20 was added to the syringe. Solvent

was allowed to drain, and additional Sephadex LH-20 was added

until the column was packed to the 1.5 ml calibration of the

syringe (approximately .5 g dry Sephadex LI--20 per column).

The syringe inner wall was washed down with solvent, and a

filter disc was placed on top of the Sephadex LII-20 bed.

Dried concentrated extracts were redissolved in .1 ml of

benzene:methanol (90:10) and transferred to the columns.

Sample solvent was allowed to enter the columns before two

washings of .2 ml each were added from the sample tube.

After addition of the sample and washings, four fractions











(void, estrone, void, estradiol) were separated by adding

known volumes (.1, 1.4, .2, 2.4 ml) of solvent in sequence

to the top of the column. This procedure has been described

and a typical elution pattern shown by Chenault et al. (1975).

Eluent from each addition was collected into separate 10 x 75

mm disposable culture tubes. The tubes containing estrone

and estradiol were dried at 45C under air. The dried extract

was redissolved in 1 or 2 ml of fresh elution solvent. A one-

tenth aliquot was removed for assessing procedural losses of

the tracer steroid, and the remaining sample was either

assayed in its entirety, split into three aliquots and assayed,

or further diluted before splitting into three aliquots and

assayed. The amount of dilution required depended upon the

steroid assayed, sample source and stage of gestation. The

estradiol antibody used in this study was a gift of V. L.

Estergreen of Washington State University.

Crossreactivity of the estradiol antibody used was

reported by Chenault et al. (1975). Estrone and estradiol-17

were the only steroids of those tested which showed any major

crossreactivity (30 and 17 percent, respectively) with the

antibody. Corticosterone crossreacted at 4 percent whereas

all other steroids tested (cortisol, estriol, progesterone,

20--OH progesterone, testosterone and androstendione) failed

to show crossreactivity.

Validation of the estrone assay is shown in Table 1.

Known concentrations of estrone were added to plasma from a










bilaterally hysterectomized-ovariectomized gilt. A quanti-

tative recovery of estrone was obtained.



Table 1. Validation of Estrone Assaya


Amount Added Amount Measured
(pg) (pg) n S.E.

10 9.8 5 2.0
20 13.9 4 2.0
50 46.3 5 6.3
100 96.6 5 9.5
150 167.7 3 14.3


aEstrone measured in peripheral plasma
from a bilaterally ovariectomized gilt.
Estimates were corrected for endogenous
estrone level in the bilaterally ovariec-
tomized gilt plasma and for procedural losses.
Y = -.6586 + 1.103X
Coefficient of variation = 24.97%



Statistical Analysis

Least squares analysis of variance procedures (Harvey,

1960) were used to analyze the data. Data were analyzed in

terms of "gilt data" and "fetal data." Gilt data analysis

involved (1) those variables which applied to the entire

litter (for example, total CL number, uterine surface area

and all steroid hormone values); (2) the sum of the various

fetal responses for the litter (for example, total allantoic

fluid volume and total placental weight); and (3) the average

of the various fetal responses for the litter (for example,

average allantoic fluid volume and average placental weight).










These data were analyzed for the effects of treatment, day of

gestation and treatment x day of gestation interaction.

Fetal data analysis involved those parameters which were

obtained on each individual fetus (for example, all fetal

fluid, placental and fetal measurements). These data were

analyzed for effects of treatment, day of gestation, treat-

ment x day of gestation interaction and gilt within treatment

x day of gestation cells. Table 2 indicates expected mean

squares in the analysis of variance of fetal data. The

residual term was used to test gilt within treatment x day of

gestation differences. Since gilts were a random effect, the

gilt variance was used to test for the effects of treatment

x day of gestation, day of gestation and treatment. Correla-

tion coefficients were determined on all possible combination

of gilt and fetal parameters. All values reported represent

least squares means + standard errors. All correlations

represent simple overall correlations unless otherwise indi-

cated.


Table 2. Analysis of Variance Expected
Mean Squares for Fetal Data


Source df Expected Mean Square
S2
Treatment (T) 1 o + K6o + K 70
e G 7T
7 2
Day of Gestation (D) 10 oe + K G + K 5

T X D 10 o2 + K 2 + K3TD
e 4 G 5 D

2 2
Gilt: T X 1) 66 o + K O
e l G
Residual N-88 o
o










Results and Discussion


General Effects

There was no significant (P>.10) treatment effect on

number of corpora lutea (CL), therefore the number of poten-

tial embryos was considered to be the same in the IC and UHOX

gilts. The average number of CL at the various stages of ges-

tation are shown in Table 3. Average ovulation rate was 12.0

in the IC gilts and 12.5 in the UIIOX gilts. This phenomenon

of compensatory ovarian function is well established (Burrows,

1949; Brinkley et al., 1964; Fenton et al., 1970). Although

ovulation rate was equal for the two treatment groups, there

were significantly (P<.01) more live fetuses (Table 3),

fewer dead fetuses (Table 4) and a greater percent fetal sur-

vival (Table 4) from day 35 to day 100 of gestation in the IC

gilts. However, differences in these parameters were not

significant (P>.10) prior to day 30 of pregnancy. Dziuk

(1968), Fenton et al. (1970) and Webel and Dziuk (1974) have

previously reported that IC and UIIOX gilts have similar repro-

ductive performance up to day 30 of pregnancy. However,

Fenton et al. (1970) reported a reduction in litter size in

UIOX gilts at 105 days of gestation to about one-half that

for IC gilts. Similarly, Webel and Dziuk (1974) found that

the reduction in uterine endometrial surface area in UIIOX

gilts resulted in increased embryonic mortality after day 30

of gestation.

Table 5 compares empt. uterine weight and length of the

uterine horn(s) in the IC and UIIOX gilts at the various stages










Table 3. Comparison of Number of Corpora
Lutea (CL) and Number of Live Embryos (LE)
in Intact Control (IC) and Unilaterally
Ilysterectomized-OvarLectomi zed (UIlOX)
Gilts at Various Stages of Gestationa



Day of No. of CL No. of LEb
Gestation IC tUHIOX IC UllOX

20 11.81.7 11.51.2 10.32.0 10.01.1
25 12.30.9 13.01.9 11.00.6 10.31.8
30 13.50.9 13.31.4 10.31.4 11.00.4
35 13.0i1.9 11.80.9 10.8+2.0 8.8+1.3
40 12.31.1 14.5+1.9 11.31.0 6.81.1
50 12.00.4 14.31.3 8.8+1.3 8.5+1.5
60 11. 8 0.3 12.01.3 10.00.4 8.00.9
70 12.511.7 12.51.2 8.511.3 5.30.7
80 11.5+1.3 12.01.1 10.5+1.2 5.80.8
90 11.31.1 12.30.9 10.3-1.2 6.50.7
100 9.52.4 10. 3 1. 1 7.3+12.1 6. 8 0.7
Overall 12.00.4 12.50.4 9.90.4 7.90.4


aAll values represent least
standard errors.


squares means


Least squares analysis of variance indicated
a highly significant (P<.01) treatment effect and
a significant (P<.05) day of gestation effect.










Table 4. Comparison of Number of Dead
Embryos (DE) and Percent Fetal Survival
(%S) in Intact Control (IC) and Uni-
laterally Hysterectomfized-Ovariectomi zed
(U1O0X) Gilts at Various Stages of Gesta-
tiona


No. of Percent
S Dead Embryosb Fetal Survivalb,c
Day of
Gestation 1C UIIOX IC UIIOX

20 0.00.0 0.00.0 85.46.4 86.42.0
25 0.30.3 0.50.5 90.96.8 79.49.5
30 1.30.7 0.80.5 75.1-6.5 82.79.2
35 0.50.5 0.80.5 80.76.4 73.77.8
40 0.0-0.0 1.80.8 92.65.5 51.814.3
50 0.30.3 2.00.8 73.511.0 60.010.2
60 0.30.3 1.80.3 85.23.9 67.15.7
70 0.30.3 3.80.9 72.313.1 42.44.4
80 0.00.0 1.5+0.7 92.05.9 50.110.3
90 0.00.0 1.00.4 '91.1+5.6 54.99.4
100 0.80.5 1.01.0 76.8+5.6 66.34.2
Overall 0.30.3 1.40.6 83.37.0 65.37.9


aAll
standard


values represent least squares means +
errors.


Least squares analysis of variance indicated
a highly significant (1P<.01) treatment effect.

CLeast squares analysis of variance indicated
a significant (P<.05) day of gestation effect.










Table 5. Comparison of Empty Uterine Weight (EUWT)
and Length of the Uterine Ilorn(s) (LUll) in Intact
Control (IC) and Unilaterally Ifysterectomized-
Ovariectomized (UilOX) Gilts at Various Stages of
Gestationa


Empty Uterine Weightb,c Length Uterine Horn(s)b
Day of (g) (cm)
Gestation IC UJIOX IC UIIOX

20 537.572.1 358.4+114.0 199.513.7 108.58.7
25 754.787.5 481.040.1 221.016.8 126.87.5
30 1174.911.0.2 631.272.4 300.023.0 128.04.3
35 1266.5172.5 832.1-60.9 250.028.8 134.36.0
40 1330.9105.1 748.4-110.3 264.817.5 112.810.3
50 1294.4+119.2 831.492.2 242.514.5 133.811.6
60 1900.9+102.5 1124.072.0 295.327.9 142.519.9
70 2125.5228.4 1058.9123.5 266.814.9 130.54.5
80 2182.7167.0 1139.2129.5 279.525.2 133.010.9
90 2266.0417.2 1231.198.9 2-76.314.8 133.76.8
100 2190.3156.4 1517.1105.2 289.325.2 141.5+14.2


aAl o
errors.


values represent least squares means + standard


Least squares analysis of variance indicated highly
significant (P<.01) treatment and day of gestation effects.
Cast squares analysis of variance indicated a
significant (P1'.05) treatment x day of gestation effect.










of gestation. Highly significant (P<.01) treatment and day

of gestation effects were found for both empty uterine weight

and length of uterine horn(s). Empty uterine weight was

found to increase steadily up to approximately 60 days of

gestation and then remained rather constant from days 60 to

100. It should also be noted that all measurements of pla-

cental growth increased very little after approximately 60

days of gestation. Highly significant (P<.01) correlation

coefficients were found between empty uterine weight and

length of uterine horn(s), percent fetal survival and all of

the placental and fetal responses measured (Tables 6 and 7).

Based upon these correlations and the pattern of uterine

weight changes observed, the data suggest that the increase

in uterine weight was due to local stimulatory effects of the

concepts, e.g., placental steroids. Highly significant

(P<.01) correlation coefficients were also found between

length of uterine horn(s) and all placental measurements, the

number of live embryos and percent fetal survival (Table 6).

However, it was not possible to determine whether this asso-

ciation was due to the uterine horn(s) being inherently long

and therefore able to support a larger number of fetuses or

due to the stimulatory effects of a large number of fetal-

placental units resulting in increased length of the uterine

horns. It is suggested that the latter possibility was

probably the case because of the production of steroids by

the placenta which stimulated uterine growth.


































































































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Table 7. Simple Correlation Coefficients
Between Uterine Surface Area (USA), Uter-
ine Surface Area per Fetus (USA/F),
lEmpty Uterine Weight (EUWT) and the
Various Placental and Fetal Measurements


USA USA/F


PLL

P LWT

PLSA


.861**

.917**

.906**


PLDVOL


TAR

CRL

FWWT


.634**

.883**

810**


.884**

.904**

860**

.853**


881**

847**


EUWT

.884**

.935**

.905**

.885**

.603**

.863**

.772**


**P<.01.


PLL = Placental Length
PLWT = Placental Weight
PLSA = Placental Surface Area
PLDVOL = Placental Displacement Volume
TAR = Total Number of Areolae
CRL = Crown Rump Length
FWWT = Fetal Wet Weight











Uterine endometrial surface area and uterine endometrial

surface area per fetus were significantly (P<.01) greater in

the IC gilts at all stages of gestation (Table 8). The

degree of reduction in uterine endometrial surface area was,

as the treatments suggest, approximately one-half. The sig-

nificance of this reduction in endometrial surface area is

apparent when one notes the highly significant correlation

coefficients between uterine endometrial surface area and the

various parameters of concepts growth (Table 7).

The protein concentrations in radial vein, uterine vein,

uterine artery and umbilical vein plasma were not significantly

(P>.10) affected by treatment or day of gestation (Tables 9

and 10).


Fetal Fluid Effects

The changes which occurred in allantoic fluid volume

during gestation are summarized in Table 11. Treatment, day

of gestation and gilt:treatment x day of gestation effects

were highly significant (P<.01). The first rapid increase in

allantoic fluid volume occurred between days 20 (IC, X = 3.8

ml) and 30 (IC, X = 209.4 ml). Then, there was a decrease in

allantoic fluid volume to day 40 (IC, X = 65.9 ml), followed

by a second increase to day 60 (IC, X = 322.6 ml) and a subse-

quent decrease to day 100 (IC, X = 64.2 ml). This general

pattern of change in allantoic fluid volume was similar for

IC and UIIOX gilts and the data support the findings of

Wislocki (1935).











Table 8. Comparison of Uterine Surface Area (USA)
and Uterine Surface Area per Fetus (USA/F) in Intact
Control (IC) and Unilaterally Ilysterectomized-
Ovaricctomized (U1IOX) Gilts at Various Stages of
Gestationa


Day of
Gestation


Uterine Surface Areab,c
(cm22 )
IC UIIOX


Uterine Surface
Area/Fetush ,d (cm2)
IC UJIOX


826. 2 111.9
1404.4112.4
2275.5258.1
2106.6-210.5
2473.9239.6
2489.9213.0
4103.5660.9
3831.6498.1
4729.8502.0
4998.5589.0
5231.9482.9


527. 353.4
815. 7 72. 7
1172.962.7
1200.0102.0
1108.3154.9
1460.9115.6
2023. 9 301. 3
1803.6+139 .3
2278. 8+272.6
2265.4126.5
3077.5243.5


84. 36. 8
129. 313.4
224.57.2
221. 346.5
220.817.6
292.820.6
414.3 70. 3
455.021.5
458.846.7
490.828.9
747.383.8


53.85.2
94.027.6
107.89.9
143.014.6
174.331.9
182. 3 25.1
257.843.1
352.3+26.3
404.5+49.5
352.017.0
458.815.0


errors


values represent least squares means standard


Least squares analysis of variance indicated highly
significant (P<.01) treatment and day of gestation effects.

Least squares analysis of variance indicated a highly
significant (P<.01) treatment x day of gestation effects.

Least squares analysis of variance indicated a
significant (P<.05) treatment x day of gestation effect.


20
25
30
35
40
50
60
70
80
90
100











Table 9. Comparison of Radial Vein Plasma Protein
Concentration (RVPRC) and Uterine Vein Plasma
Protein Concentration (UVPRC) in Intact Control
(IC) and Unilaterally flysterectomized-Ovariectomized
(UIIOX) Gilts at Various Stages of Gestationa


Day of
Gestation


RVPRC (g/ml)
IC U1IOX


UIVPRC (mpg/jnl)b
IC UJIOX


102.135.8
115.2+17.9
122. 3 12.1
108. 824.6
106. 318.9
119.314.8
115.618.8
91. 6 21. 5
125.92.2
93. 3+3.6
122.126.1


84. 0+21.6
81. 89.8
91.9--9.0
127. 6 3.6
103.5-16.6
123. 7 20.9
81.99.8
120.325.9
106.117.7
90. 1 20.6
84 .7 7.3


87.0- 21.2
118. 6 12 .4
102. 7 14. 7
107.218.4
110. 7 26 .3
128.319 .9
116.621.3
86.9+13. 9
104. 76.9
95.09.5
112.534.3


77.026.0
81.625.9
86. 88.7
135.440.9
82.217.1
139.415.8
86.9+11.0
126.4+30.3
105.09.9
99.1+28.7
82.5-16.4


aAll values represent least squares means
errors.


standard


Least squares analysis of variance indicated no
significant (P>.10) differences.


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Table 11. Comparison of Allantoic
Fluid Volume (ALVOL) in Intact Control
(IC) and Unilaterally Ilysterectomized-
Ovariectomized (UllOX) Gilts at Various
Stages of Gestationa


D Allantoic Fluid Volume (ml)C
Day of
Gestation NI IC N U11OX

20 41 3.80.3 38 3.00.3
25 44 83.6+2.7 40 56.53.7
30 41 209.46.9 44 136.29.4
35 43 100.6+9.2 27 112.311.9
40 45 65.9+3.9 27 81.511.0
50 35 169.611.5 34 127.4+16.5
60 37 322.638.9 30 279.142.7
70 32 185.131.7 21 125.230.1
80 40 100.2+12.9 22 136.421.9
90 40 76.410.3 26 72.613.6
100 29 64.212..3 27 39.7+13.0


aAll values represent least
+ standard errors.


squares means


N = Number of observations.

CLeast squares analysis of variance indicated
highly significant (P<.01) treatment, day of
gestation and gilt:treatment x day of gestation
effects.










The rapid increase in allantoic fluid volume between

days 20 and 30 of gestation was associated with, and probably

induced, initial expansion of the chorio-allantois membranes

which forced those membranes into apposition with the uterine

endometrial surface. At day 30 of gestation, highly signifi-

cant correlation coefficients were found between allantoic

fluid volume and placental length (r=.652), placental weight

(r=.736), allantoic fluid total protein (r=.498) and allantoic

fluid estrone concentration (.898). A rapid increase in

allantoic fluid estrogen concentration also occurred between

days 20 and 30 of gestation and is believed to be the primary

stimulus for the initial allantoic fluid volume increase.

The final period of expansion of the placenta occurred

between days 40 and 60 of pregnancy.. At day 60 of gestation,

which coincided with the second peak and maximum volume of

allantoic fluid, the correlation coefficients between allan-

toic fluid volume and placental length, placental weight, and

allantoic fluid total protein were .571, .596 and .734,

respectively. These correlation coefficients were also highly

significant (P<.01). The overall simple correlation coeffi-

cients (based upon 768 observations) between allantoic fluid

volume and placental length (r=.374), placental weight

(r=.284) and allantoic fluid protein (r=.735) were also highly

significant (P<.01). The most obvious reason for the corre-

lation coefficients between allantoic fluid volume and pla-

cental length and weight being lower when all stages of ges-

tation were considered is tre fact that placental length and











weight, once established, do not decrease, while allantoic

fluid volume undergoes two periods of rapid increase and

decrease.

The degree to which the chorio-allantois membranes are

expanded by the allantoic fluid is of fundamental physiologi-

cal importance because this determines the maximum placental

surface area, the maximum number of uterine glands the pla-

centa comes into contact with, and therefore the maximum

number of placental arcolae. The placental areolae differen-

tiate only where the chorio-allantois lies in direct apposi-

tion to the opening of a uterine gland onto the endometrial

surface (Brambel, 1933). Several workers have suggested that

the areolae serve specifically as sites of absorption and

transport of secretions of the uterine glands (von Baer, 1828;

Turner, 1875; Goldstein, 1926; Brambel, 1933). More recently,

Chen et al. (1975) utilized immunofluorescent antibody tech-

niques to demonstrate that the uterine specific purple acid

phosphatase in pigs is secreted by the uterine endometrial

glands, transported across the chorio-allantois membranes via

the areolae and sequestered in the allantoic fluid.

The changes which occurred in allantoic fluid protein

concentration and allantoic fluid total protein are summarized

in Table 12. Protein concentration (P<.05) and total protein

(P<.01) were significantly greater in the IC gilts. Day of

gestation and gilt:treatment x day effects were also highly

significant (P<.01) for both variables. There was a rapid































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accumulation of protein in the allantoic sac until day 60 of

gestation and a rapid decline from days 60 to 100.

The data suggest that the osmotic gradient set up in the

allantoic sac by the rapid accumulation of protein therein

between 40 and 60 days of gestation may have resulted in

increased water movement in association with the increased

protein movement into the allantois and thus may be respon-

sible for the second rise in allantoic fluid volume which

reached a peak at day 60 of gestation. It is further sug-

gested that the rapid decrease in allantoic fluid volume and

allantoic fluid total protein between days 60 and 100 of ges-

tation may be due to the rapid increase in estrogens and

decline in progesterone during this period. Knight et al.

(1974b) indicated a negative relationship between estrogen

levels and the quantity of uterine protein recovered when

the estrogen levels were given in excess of that necessary

for a synergistic relationship with progesterone. The possi-

bility exists that the high plasma estrogen levels from days

70 to 100 of pregnancy may inhibit protein synthesis by the

uterine glands. As a consequence, there is a lack of protein

moving into the allantoic sac, the osmotic gradient necessary

to stimulate water movement declines and finally allantoic

fluid volume declines.

The pattern of amniotic fluid volume changes during ges-

tation are summarized in Table 13. Measureable amounts of

amniotic fluid were not present prior to day 30 of gestation.

In the IC gilts, amniotic I luid volume increased from day 30










'Fable 13. Comparison of Amniotic Fluid
Volume (AMVOL) in Intact Control (IC)
and Unilaterally Ilysterectomi zed-
Ovariectomized (UllOX) Gilts at Various
Stages of Gestationa


Dy o Amniotic Fluid Volume (ml)d
Day of
Gestationb Nc IC N UIIOX

30 41 2.00.1 43 1.9-0.1
35 42 5. 7 0.2 34 5.30.3
40 45 11.210.3 27 10.40.4
50 35 41.92.31 34 28.7+1.5
60 39 105.23.3 30 73.95.2
70 32 196.2 112.8 21 110.48.5
80 40 193.5+13.3 22 170.420.2
90 41 118.38.4 26 137.418.3
100 29 117.37.5 27 85.211.9


aAll values represent least squares means
standard errors.

bNo measurable amniotic fluid was present
prior to day 30 of gestation.

CN = number of observations.

Least squares analysis of variance indi-
cated highly significant (P<.01) treatment,
day of gestation and gilt:treatment x day of
gestation effects.










to day 70, plateaued to day 80 and decreased to day 100. In

the UIOX gilts there was a steady increase to day 80 and a

subsequent decrease between days 80 and 100. There were

highly significant (P<.01) treatment, day of gestation and

gilt:treatment x day effects. The changes in amniotic fluid

volume observed in this study are in general agreement with

those reported by Wislocki (1935).

Amniotic fluid protein concentration and amniotic fluid

total protein changes during gestation are summarized in

Table 14. In both IC and UIIOX gilts, maximum amniotic fluid

protein concentration was reached on day 60 of gestation and

maximum amniotic fluid total protein on day 70 of gestation.

There was a steady decrease in both treatment groups from

these maximum values to day 100. There was no significant

(P>.10) treatment effect on amniotic fluid protein concentra-

tion, but amniotic fluid total protein was significantly

(P<.01) greater in the IC gilts due to the larger amniotic

fluid volume. Total protein in amniotic fluid was less than

total protein in allantoic fluid at all stages of gestation.


Placental and Fetal Effects

Placental length was significantly (P<.01) greater in

the IC gilts at all stages of gestation (Table 15). As early

as day 30, average placental length was 13.5 cm greater in

IC (49.6 cm) compared with UHIOX (36.1 cm) gilts. There was

a rapid increase in placental length between days 20 (IC, X =

7.3 cm) and 30 (1C, X = 4(9. cm) of gestation. As previously
































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Table 15. Comparison of Placental Length
(PLL) in Intact Control (IC) and Uni-
laterally lHysterectomi zed-Ovariectomized
(UIIOX) Gilts at Various Stages of Gesta-
tiona



Day of Placental Length (cm)c
Gestation Nb IC N UIIOX

20 41 7.30.3 40 6.50.4
25 44 33.0+0.8 41 22.81.0
30 41 49.6-1.6 44 36.11.5
35 43 47.41.8 35 43.82.4
40 45 53.21.8 27 46.42.7
50 35 62.12.0 34 49.32.3
60 40 74.812.3 32 56.8+2.7
70 34 83.73.3 22 62.54.2
80 42 75. 1 1.7 24 63.53.0
90 41 75.6+2.6 26 64.73.5
100 29 88.6+3.0 27 68.91.9


aAll values represent least
+ standard errors.

N = number of observations.


squares means


CLeast squares analysis of variance indi-
cated highly significant (P<.01) treatment,
day of gestation and gilt: treatment x day
of gestation effects.










discussed, this early rapid increase in placental length was

coincidental with and probably induced by an equally rapid

increase in allantoic fluid volume during the same period of

gestation. Placental length continued to increase to day 60

of pregnancy, but changed relatively little thereafter.

Warwick (1928) and Pomeroy (1960) also reported that the pig

placenta attained its maximum size by approximately day 60 of

gestation and grew little, if any, thereafter.

Placental weight was also significantly (P<.01) greater

in the IC gilts at all stages of gestation (Table 16). At

day 30, there was an average difference of 10.9 g in placental

weight between IC (27.6 g) and UIIOX (16.7 g) gilts and, by day

100, this difference had increased to 63.5 g (258.0 vs. 194.5

g). As observed earlier by Warwick (1928), the increase in

placental length preceded the increase in placental weight and

placental weight changed little after day 60 of gestation.

Since the placenta grows relatively little after day 60 of

gestation, fetal growth in the latter stages of gestation,

which is the period of maximum fetal growth, is largely depen-

dent upon the extent of placental development which occurred

during the first 60 days of pregnancy. It is further appar-

ent that under conditions of intra-uterine crowding the extent

of placental development is limited. Therefore, it is sug-

gested that the highly significant (P<.01) decrease in fetal

growth and increase in fetal mortality which occurred in the

UHIOX gilts during the latter stages of gestation were due to











Table 16. Comparison of Placental Weight
(PLWT) of Intact Control (IC) and Uni-
laterally Ilyste rectomi zed-Ovariectomized
Gilts at Various Stages of Gestationa



Day of Placental Weight (g)C
Gestation Nb IC N UIIOX

20 41 0.210.02 40 0.180.02
25 44 8.3-0.5 41 5.30.5
30 41 27.61.1 44 16.71.1
35 43 49.12.9 35 35.42.3
40 45 54.22.4 27 40.93.0
50 35 106.3+4.8 34 57.56.4
60 40 174.27.7 32 119.112.4
70 34 240.714.0 22 157.3+17.5
80 42 178.8-6.6 24 153.6+10.9
90 41 208.611.3 26 159.411.8
100 29 258.019.3 27 194.59.3


All values represent least
standard errors.


squares means


bN = number of observations.

CLeast squares analysis of variance indicated
highly significant (P..01) treatment, day of
gestation and gilt:treatment x day of gestation
effects. Treatment x day of gestation was sig-
nificant (P<.05).











placental insufficiency. The smaller placental mass of the

UIIOX gilts was simply incapable of supporting continued

fetal growth.

Placental surface area and placental displacement volume

were also significantly (P<.01) greater in the IC gilts at

all stages of gestation (Tables 17 and 18). Both of these

parameters changed relatively little after day 70 of gesta-

tion. Highly significant (P<.01) correlation coefficients

were found between all placental parameters measured (Table

19).

As observed by Brambel (1933), the areolae appeared

initially in greatest concentration in the interior sections

of the placenta and then developed toward the polar sections

so that by day 50 of gestation there was no significant

(P>.10) difference in the number of areolae between the two

areas of the placentae (Table 20). There were significantly

(P<.01) more areolae in both the interior and polar sections

of the placenta in the IC vs. UHOX gilts. Areolae surface

area was also significantly (P<.01) greater in both the

interior and polar sections of the placenta in the IC gilts

(Table 21). Total number of areolae per placenta and total

areolae surface area were also significantly (P<.01) greater

in the IC gilts (Table 22). Day of gestation and gilt:treat-

ment x day effects were highly significant (P<.01) for all

areolae responses. Brambel (1933) estimated that the number

of areolae reached a maximum at about 35 to 40 days of gesta-

tion. Data from this study indicated the number of areolae











Table 17. Comparison of Placental Surface
Area (PLSA) in Intact Control (IC) and
Unilaterally Hysterectomized-Ovariectomized


(UIIOX) Gilts at Various Stages


of Gestation"


y o Placental Surface Area (cm2 )
Day of
Gestationb Nc IC N UIIOX




All values represent least
standard errors.


squares means


bNo measurements were taken prior to day 35 of
gestation.

CN = number of observations.

Least squares analysis of variance indicated
highly significant (P<.01) treatment, day of
gestation and gilt:treatment x day of gestation
effects.


I











Table 18. Comparison of Placental Displace-
ment Volume (PLDVOL) in Intact Control (IC)
and Unilaterally IIysterectomized-Ovariec-
tomized (IIUOX) Gilts at Various Stages of
Gestationa


Day of
Ge stati on0)


Placental Displacement


Volume (ml)d
U11JOX


42 40.82.2
43 51.93.2
35 97. 0 3.5
35 153.28.9
31 213.6+12.5
41 151.46.1
30 154.78.3
28 208.7+8.6


aAll values represent least
standard errors.

bNo measurements were taken
of gestation.


34 33.512.3
23 41. 7 3.5
33 60.46.5
32 113.211.8
17 143.221.5
24 138.9+9.2
24 145.810.0
27 157.39.2


squares means +


prior to day 35


cN = number of observations.

Least squares analysis of variance indicated
highly significant (P<.01) treatment, day of
gestation and gilt:treatment x day of gestation
effects.


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