GROWTH AND DEVELOPMENT OF THE SWINE FETUS

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
JAMES I. SPRAGUE Jr.
1961

This is to certify that the

thesis entitled

Growth and Development of the Swine Fetus

presented bg

James I. Sprague, Jr.

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Animal HUSbandl‘y

D. E Ullrey

Major professor

 

Date May ‘9, 1961

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ABSTRACT

GROVE AND DEVELOPMENT OF THE SHINE FETUS

I. Measures of body size, organ
weights and skeletal development.

II. Time of appearance and quantitative

estimation of intestinal lactase and
alkaline and acid phosphatase.

by James I. Sprague, Jr.

The objectives of the study were to provide information
hitwo areas concerned with the development of the swine fe-
tus;

1. Measures of body size, organ weight and skele-
tal development which would promote better understanding of
the relationships between the anatomy and physiology of grow-
ing fetal structures and which would allow for more accurate
estimation of age of fetuses of unknown conception date.

2. Estimation of the time of appearance, location
and concentration of intestinal lactase and acid and alkaline
phosphatase.

Fetal and newborn pigs were obtained from 19 first-litter
Yorkshire gilts which were selected for uniformity of size
”“1 a‘80- Supplementary information was gathered from fetuses

from 7 Hampshire 1 Duroc gilts and piglets from it Duroc sec-

Gad-litter sows. At appropriate estrous periods, the gilts

4 _—-ro——-—-—.d—_——

James I. Sprague, Jr.

were bred to 2 Yorkshire boars, 1 serving a gilt once on the
1st day followed by the 2nd bear on the succeeding day. If
further estrus were not observed, the gilts were considered
pregnant and caesarian sections were performed 30, 51, 72 or
93 days post-breeding or the gilts were allowed to farrow
naturally. The crossbred gilts were slaughtered at 15 days
and the fetuses removed from the excised uteri.

Body weights, crown-rump lengths and head widths across
the parietal bones were measured immediately after removal
from the uterus or Just following birth. The weights of in-
ternal organs and the lengths of the humeri were obtained
immediately subsequent to dissection. The other length
measurements were obtained from X-ray photographs and were
measures of the calcified diaphyses of the bones of the
appendages.

Sections of the gastro-intestinal tract were separated,
identified and frozen in dry ice where they were stored un-
til enzyme assays could be performed.

Correlations of litter size with measures of fetal
Browth were accomplished at each fetal period. Size of lit-
ter did not consistently effect the dimension of any parti-
cular measure. The data, however, did reveal a smaller pro-
POrtion of negative correlations for measure versus litter
313° at 72 days following breeding than in any other fetal
period studied or at birth.

Although the differences for most measures between males

and females were generally not significant, male fetuses and

 

 

 

James I. Sprague, Jr.

male piglets were slightly heavier, longer and the organs
heavier at each of the periods.

Means, standard errors of the means and relative stand-
ard errors of the means for both sexes and for the sexes
combined were presented. Skeletal measures gave smaller
relative standard errors than measures of soft tissue or
weight of the whole fetus.

Growth patterns plotted from various measurement means
cfi'fetal and newborn pigs were presented. Measures of
skeleton were generally linear from.51 days to birth. The
adrenals made the majority of their growth the last 3 weeks
of fetal life.

Correlations of measures of fetal growth with age post-
breeding were highly significant. Coefficients of age cor-
related with measures of skeleton ranged above 0.95 except
for head width, while measures of soft tissue ranged from
0.6!; to 0.90.

Five estimating equations were suggested for use in
predicting ages of fetuses from.Yorkshire first-litter gilts.
The particular measures selected met several important cri-
teria, which were that they: (1) be easily measured, (2) be
consistently duplicated, (3) be highly correlated with fatal
‘80 and (h) possess a low relative standard error of the
mean ateach fetal age. Standard errors of estimate of the
Predicting equations were all below $1.0 day.

At birth, the middle portion of the small intestine

possessed a greater enzymatic activity for lactase and alkaline

James I. Sprague, Jr.

and acid phosphatase than did the cranial or caudal portions.
Acid phosphatase was more uniformly distributed throughout
the small intestine than either lactase or alkaline phospha-
tase except in the cranial duodenum where its activity was
lower.

Activity of these 3 enzymes were significantly greater
at birth than at any of the fetal periods studied. Alkaline
phosphatase activity was also significantly higher at 93 days

than at 72 days.
Time patterns for the appearance of alkaline and acid

phosphatase which had been reported for other fetal species
were confirmed for the fetal pig.

GROWTH AND DEVELOPMENT OF THE SHINE FETUS

1. Measures of body size, organ
weights and skeletal development.

II. Time of appearance and quantitative
estimation of intestinal lactase and
alkaline and acid phosphatase.

BY

James I. Sprague, Jr.

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Animal Husbandry

1961

ACKNOWLEDGMENTS

The author wishes to express his appreciation to the
people of Hichigan.and to Michigan State University for the
opportunity to study and do research at that institution.
Sincere Appreciation is extended to Dr. Ronald Nelson, Pro-
fessor, and Head of Animal Husbandry for award of the Gradu-
ate Research Assistantship and provision of the facilities
fbr conducting the study; to Drs. Duane Ullrey and Elwyn
Miller,.Assistant Professors of Animal Husbandry for their
guidance, counsel and continued interest throughout the
study; and to the members of his guidance committee, Dr.
Jacob Hoefer, Professor of Animal Husbandry, Dr. Paul
Heineke, Professor of Physiology, Dr. Richard Byerrum, Pro-
fhssor of Chemistry and Assistant Provost and Dr. Gabel
Conner, Professor of Surgery and Medicine for their guid-
ance and counsel. The efforts of Dr. William Magee, Assoc-
iate Professor of Animal Husbandry, in providing advice on
the statistical analyses are much appreciated.

The author 13 also extremely indebted to his Graduate
Research Assistant colleagues who gave their time and as-
sistance.‘ They are Drs. Waddill, Sutton, Pratt, Hoersch,
3°°Pley,1Heaney,'YOung and Makarechian and Messrs. Harmon,
Ritchie, Hines and Pollard. Also sincere gratitude 18 ex-
tended to Mrs. Eleanor Salmon.Alexander and Mr. Chesley

Zutaut for assistance in collecting the data, to Dr. Hello

11

Van Pelt, Research Associate of the Department of Surgery
and Medicine, for performing the caesarian sections, to Dr.
U. V. Hostosky, Instructor of the Department of Surgery and
Medicine, for performing the radiology of the fetal and
newborn pigs, and to Mr. G. B. Stafford, Swine Herdsman for
care of the sows and gilts.

The author wishes to express his gratitude and apprec-
iation to his wife Jeanne for her sacrifices, understanding
and encouragement, and also to his sons Anthony John and
James Stuart for their sacrifices.

Finally, the author would like to thank his parents
for encouraging pursuit of knowledge all during his life,
to his former colleague V. C. Beal for the encouragement
of the authors professional training and especially to Dr.
Duane Ullrey, the author's major professor for his hand
of encouragement, his gift of patience and his teaching of
precision and desire for seeking the facets. of truth through

research.

’ iii

TABLE OF CONTENTS

ImmDUCTION O O O O O O O O O 0
REVIEW OF LITERATURE . . . . . .

1. Body size, organ weight and skeletal
development . . . . . . . .

Partition Of nutrients e e e e e e

Heritability of traits associated with
reproductive efficiency of swine . . .

Factors affecting fetal weight . . . .

Early fiPOSHancy e eeeee ee eee

Studies of fetal development in swine

2. Fetal nutrient deposition as a measure
of food requirement of pregnancy in swine

3. Development.of intestinal carbohydrases

and acid.and.alkaline phosphatase in

the pig

Neonatal and.fetal development of the
intestinal carbohydrases of the pig

alkaline and

Occurrence of intestinal

acid phosphatase . . .
MATERIALS AND METHODS . . . . .
EXperhmental subjects . . .
Feeding . . . . . . . . .
Breeding . . . . . . . . .
Surgical.procedure . . . .
Manipulation of the fetuses
Tissue for enzyme analysis

X-ray of the fetuses

O

and

O

newborn

pigs

Organ dissectionpandworgan.weight . . . . . .

iv

Page

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25

29
29

37

In
In
#3
#3
#3

#5
#5

Preparation of tissues for enzyme analysis
Lactase assay procedure . . . . . . . . .
Procedure for alkaline phosphatase . . . .
Procedure for acid phosphatase . . . . . .
RESULTS AND DISCUSSION . . . . . . . . . . . .
Effect of litter size on fetal growth . .
Effect of sex of the fetus on fetal growth
Effect of fetal age on fetal growth . . .
Correlation of measures with fetal age . .
Predicting fetal age from fetal growth data
Estimation of time of appearance, location

and concentration of intestinal lactase and
alkaline and acid.phosphatase . . . . . .

SIIWYCOOOOOOOOOOOOOOOOOOOO

LITERATURE C 1m 0 O O O O O O O O O O O O O O O

APPmIXTABLESO0000000000000...

Page
#7
hB
#9
So
52
52
55
57
68

71

73

77
81

89

Table
l.
2.

3b.
h.
5.

9.
10.
11.

12.
13.

LIST OF TABLES

Analysis of reasons for slaughter of sows .

Percent degenerate fetuses . . . . . . . .

Ration l - self-fed to Yerkshire gilts . .

Ration 2 - hand-fed to Duroc sows . . . . ..

Measures of fetal growth . . . . . . . . .

Correlation coefficients of litter size
with measures of fetal growth . . . . . . .

Percentage negative correlations of litter
size with measures of fetal growth . . . .

Mean litter size and variability of litter

8129 e e e e e e e e e e e e e e e e e e e

Effect of sex of the fetus on fetal growth
atw5 fetal.periods - t values of males
VQPSUI ramal.8» e e e e e e e e e e e e e e

(9a through 9h). Comparison of relative
standard error or the mean e e e e e e e e

Fetal growth - Yorkshire first-litter

gilts O O O O O O O O O O O O O O O O O O 0

Correlations of age with measurements of
fatal growth e e e e e e e e e e e e e e e

Enzyme distribution - birth . . . . . . . .

Fetal development of lactase, alkaline and
acid phosphatase activity . . . . . . . . .

vi

Page
. 10
0 2h
- #2
- #2
. ##
. 53
. Sh
0 SS
. 56
60.61.62
. 63
. 69
. 7h
. 7h

LIST OF APPENDIX TABLES

Table Page

1. DevelOpment of the fetal pig - measures
at birth (Data from first-litter Yorkshire

311t8)eeeeeeeeeeeeeeeeeeee

2. Development of the fetal pig - measures
at birth (Data from second-litter Duroc

80"8)eeeeeeeeeeeeeeeee

89

3. Development of the fetal pig - measures
at birth (Combined data from Yorkshire

gilts and 2nd-litter Duroc sows) . . . . . . 91,92

1;. Development of the fetal pig - 93 days

(Data from first-litter Yorkshire gilts) . . 93.914

5. Developmmt of the fetal pig - 72 days

(Data from first-litter Yorkshire gilts) . . 95,96

6. Development of the fetal pig - 51 days
(Data from first-litter Yorkshire gilts) . . . 97,98

7. Development of the fetal pig - 16 days
(Data from crossbred first-litter gilts) . . . 99,100

8. Development of the fetal pig - 30 days
(Data from first-litter Yorkshire gilts) . . . 101

9.Enzymeanalysis-birth...........102
10. Enzyme analysis-93days .......... 103
11.Enzymeanalysis-72days..........10h
12. Enzyme analysis - 51 days and 30 days . . . . 105

13. Percent dry matter of fetal intestinal

tissue ...........o........105

vii

LIST OF FIGURES

Figure Page

1 through 7e
1. Height, crown-rump length, head width . . . 6S

eeeee 65

Growth patterns of Yorkshire fetal pigs

2. Brain, liver, lung . . . . . . .

3. JHumerus, ulna, radius, metacarpal (3 or
hand20r5)............... 66
I4. Femur, tibia, fibula, ilium, ischium,
66

calcaneous, metatarsal (3 or I; and 2 or 5).

5. Heart, humerus (tuberosity to condyle),

humerus (head to condyle) and left and
rightkidneys eeeeeeeeeeeeee 67
6. Spleen, left and right gonad of the male, 6
. 7

left and right.gonad of the female . . . .
7. Thyroid, left and right adrenal and

pituitary eeeeeeeeeeeeeeee 6?

viii

INTRODUCTION

Technology often stimulates bold changes in current
swine management practices. For instance, if the lactation
pmriod of a sow could be shortened, additional nutrients
now fed the sow during gestation could be eliminated. Ad-
vances such as the development of satisfactory milk replaco
ers, excellent baby pig creep rations, as well as control of
tmby pig diseases have made considerable progress toward
weaning of piglets at one month or earlier. If these prac-
tices are made feasible, then the sow will not have to be
fed during pregnancy for the ensuing lactation period. As
a consequence, nutrients fed during late pregnancy could be
reduced to a large extent and would be used primarily for
the purpose of fetal development. »

If the sow does not have to be fed for lactation, but
only for fetal development, then nutrition studies of swine
pregnancy could place more emphasis on the fetus.

Is it possible to study swine nutrition from the view-
point of the fetus? Early research of Mitchell g£_§l. (1931)
and current research of De Villiers gt_gl. (1958) have accompe
lished this objective to some degree. However, their studies
were only steps toward complete understanding of the nutrit-
ional requirements of the fetal pig.

The following study was initiated to add information

to this now rapidly developing area of research. The

- 1 -

-2-

pmrposes were not only directed by practical problems of
swine husbandry, but by interest in their fundamental bio-
logical implications. It was.hoped that this study would

provide a few more facts for a better understanding of one
of natusz more miraculous events - growth and development

of the fetus .
The objectives of this study were to provide addition-

al information in two areas:

1. Measures of body size, organ weight and skele-
tal development which would promote a better
understanding of the relationships between the
anatomy and physiology of growing fetal struc-
tures and which would allow for more accurate
estimation of age of fetuses of unknown con-
ception date.

2. Estimation of the time of appearance, location

and concentration of intestinal lactase and

acid and alkaline phosphatase.

REVIEW OF LITERATURE

1. Body size, organ weight and skeletal deveIOpment.

Partition of nutrients

Hamond (191th) postulated that the fetus has a prior-
ity for nutrients. This concept has been termed by. Robin-
son (Hanmond, 1957) the "Theory of Partition of Nutrients.”
The theory was based on the belief that the nutritional re-
quirements of various tissues of the maternal animal and
the fetus were governed by the metabolic rates of these tis-
sues at different stages of development. Hammond suggested
that as pregnancy proceeds, competition between the fetus
and certain maternal tissues becomes greater, caused in
Part by decreased fetal metabolic rate.

Barcroft (1913.6) tested the theory proposed by Hammond.
Using the sheep as an experimental animal, he found, as
Hammond postulated, that fetal tissues had higher metabolic
rates than their maternal counterparts with the exception
of muscle and lung. On a dry weight basis, the differences
were even greater. However, contrary to Hammond's theory
there was not a decrease in metabolic rate of the fetus fol-
lowing the 99th day to term (150 days gestation period in
sheep). On a wet tissue basis, Barcroft found an increase
in metabolic rate during this period.

Wallace (l9h8) found fetal tissues, particularly ner-

vous tissue and skeleton, were less effected by low

-3-

-h-

nutritional level than other tissue. Carlyle (l9h5) observ-
ed, as did Wallace that not all fetal tissues develop at the
same rate. Hallace found the following order of organ
weight loss relative to the weight of the fetus as a whole
in ewes on a low plane of nutrition in late pregnancy:

1. Loss proportionately greater than the whole fetus-

liver
fat

2. Loss preportionately to the whole fetus-
blood vessels
muscle
lungs

3. Loss proportionately less than the whole fetus-
alimmntary tract
bone
heart
pancreas
tongue

eyes
nervous tissue

Information on metabolic rates of various fetal tis-
sues at all stages of pregnancy and on several planes of
nutrition is considered important in determining nutrition-
al requirements for the sow and for the nutrition of the
fetus during development. According to Robinson (Hammond,
1957) "This whole concept is extremely important as it lays
°mPhasis on the probability that the fetus can exercise a
demand and is not entirely at the mercy of its environment."

The work of Hammond, Barcroft, Wallace and Carlyle
'“SSested that differences in fetal growth occurring at
different periods of fetal development were caused in part

9? differences in metabolic rates and subsequent partition

of nutients.

Heritability of traits associated with reproductive

I

ggficiency of swine
The heritability of traits which measure efficiency of
swine reproduction.was studied by Cummings g§_gl. (l9h7).
They found the following heritabilities:

Survival from birth to weaning 'uofl
Size of litter at birth 22%
Total litter weight at birth 36%

Size of litter at weaning 3
Total weaning weight of the litter 7%

These heritability percentages indirectly suggested
the importance of proper environment during both gestation
and lactation for economical swine production. Since herita-
bility of litter size and litter weight at birth was quite
low, the research worker has a productive area for invest-
igation with promise of important economic advantage as a
result of new findings.

Factors affecti fetal wei t

Waldorf 22_§l. (1958) studied factors affecting fetal
P18‘Height late in gestation. They found in gilts that age
of dam appeared responsible for a greater part of vari-
ability in fetus weights than dam's carcass weight or back
fat thickness. Condition of the sow, as measured by carcass
weight and back fat thickness, did not account for a signi-
ficant variation in fetus or membrane weights.

In~mfik1ng reciprocal Shetland and Shire crosses in
horses and studying the birth weight and placenta weight,

Hammond (19hh) noted a marked decrease in the size of the

-6-

foal from the Shetland dam. Hammond indicated the limita-
tions of the higher rate of the metabolism of the maternal
tissues in the small breed than in the large may have been
the cause of the decrease in birth weight. Another possible
explanation for the decrease in birth weight was a limit of
special growth substances of maternal origin.

Hammond (1932) also had shown that single lambs were
28 percent heavier than twins. ‘dishart and Hammond (1933)
noted rabbits with 8 to ll young per litter averaged 1&5
grams at birth while in litters of l to 2 the weight was
95 grams at birth.

It was therefore evident that size of the litter had
an important effect on the growth of the fetus. In swine,
this was noted by Mitchell M. (1931) and D8 Villiers

at do (1958) 0

Early pregnancy
McKenzie (l9h8) emphasized the importance of good

 

nutrition of the gilt and sow during early pregnancy by
noting a close relationship between gains in weight of the

sow during the 11. weeks following breeding and the number

or pigs farrowed.

Self et a1. (1955) compared full versus slightly limit-

ed rations for Chester Whites and Poland China gilts. He

found full feeding during puberty (during the time between

first and second estrus) and for 25 days past the second

estrus resulted in the most number of ova shed. However:

the limited fed group had a greater survival of embryos.

-7-

These workers then found the greatest overall efficiency
combined the 2 systems. This combination involved limited
feeding during the pro-pubertal period, full feeding during
the estrous period (the gilts were bred at 2nd estrus)
and limited feeding from breeding until the 25th day had
elapsed. Self pointed out this data was in agreement with
that of Robertson 2331. (1951) and Christian and Nofziger
(1952) who reported higher ovulation rates and lower embryo
survival rates with a “high” level of feeding than with a
"moderate" feeding system during the period after breeding.

Haines 1131. (1959) in an experiment designed to study
the effect of energy intake, found full feeding Duroc-Jersey
gilts resulted in more ova shed. The full fed gilts and the
limited fed gilts were equal in their fertilization rate in
his experiment.

Studies of fetal development in swine

Ullrey in 195).). reviewed the early research including
the work of Kiebel (1897), Lowrey (1911) and Warwick (1928).
Ullrey pointed out that the work of Kiebel is now of limit-
ed value because of changes of breeds, nutrition and type.
The usefulness of Lowrey's and Warwick's research is also
somewhat limited for these reasons and because sows at that
time did not receive gestation rations fortified with trace
elements and vitamins as rations are formulated today. Lowrey
and Warwick recovered their fetal pigs from slaughter houses
and the length of gestation was estimated from crown-rump

measures. Lowrey's research was particularly interesting

-8-

because of his method of reporting the growth of the fetus.
He expressed measures of fetal structure as a percentage of
body weight.

”Warwick (1928) found fetal weight increased most rapidly
during the last 20 days of gestation than earlier while fetal
length increased at a nearly uniform rate during the last
20 days of gestation. The variation in fetal weight increas-
ed as pregnancy progressed and as litter size became larger.
Warwick proposed that the average of all normal subjects
rather than measurements of any one individual would give the
best estimate of fetal age.

Ullrey (19514) gathered data concerning relative size
and development of fetuses removed from sows and gilts. He
prepared a "normal" standard of organ weights and skeletal
measures and was able to improve the accuracy with which
the age of the fetus of unknown conception date could be
estimated. The standards also served as a basis for compar-
ing measurements from abnormal fetuses.

Ullrey used the method of least squares to reduce his
data to quadratic and cubic polynomials. He described dif-
ferent equations for first-litter gilts and-second litter
sows for each fetal measurement. The organ and tissue meas-
ures evaluated were weight of body, brain, heart, lung, 'liver
and kidney. X-ray measures were performed of the calcified
diaphysis of the humerus, radius, ulna, femur and tibia.

Gross measurements included, crown-rump length, head width

and dissected humerus length.

-9-

Ullrey proposed that it would be desirable to have a
single organ or bone which in cases of edema and partial re-
sorption would best represent the growth of the fetus. He
found the dissected humerus would satisfactorily meet this
objective.

Newland (1955) and Newland and Davis (1961), in a study
primarily designed to measure fetal uptake of manganese in
swine, observed several factors influencing fetal growth.
Newland's observations are summarized below:

(1) Normal subjects were born from low manganese
gestation rations containing 6 to 100 parts per million of
umnganese. Also no effects on fetal weight or phosphorus
metabolism were noted from these low manganese diets.

(2).A negative correlation was found between the
rmmber of fetuses in the litter and fetal weight.

(3). Radiophosphorus absorption and concentration
cfi'inorganic phosphorus was directly proportional to the
weight of the fetus.

(h) In early gestation small fetuses had a faster
uPtake of P32 and higher specific activity than larger fe-
tuses.

(5) No significant difference between weight and
uterine position was noted in 80 and 110 day fetuses; how-
ever, a pattern was noted for 65 day fetuses which indicated
the center fetuses in the horn were lighter than at the apex
or bifurcation.

Further studies of fetal growth and development were

 

 

 

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-10-

described by Pomeroy (1960 a,b,c,d,) in his monumental thesis,
“Infertility and neonatal mortality in the sow." A compre-
hensive review of Pomeroy's work follows:
Reasons for disposal of sows

Pomeroy (1960a) from a field survey, found that the
two most important reasons for disposal of sows were repro-
ductive failure and piglet mortality. He states, "The latter
was known to be a serious problem in commercial pig produc-
tion, but the short breeding life of sows and the fact that
a considerable proportion of them become sterile after only
one or two litters does not appear to have been generally
recognized." The reasons for slaughtering sows and the per-
centage of the total as analyzed by Pomeroy are listed in

Table 1.

Table 1. Analysis of reasons for slaughtering sows.
(Pomgroy, 1960a)

Henson for slaughter

 

Failure to breed 21 21.3
Piglet mortality 17 17-
Old age 150 15.0
Low fertility 101 10.1
Disease 75 7-5
Kill: failure and udder troubles 61 6.1
Uneven or unthrifty litters (+6 4-6
Foot and Mouth disease restrictions 32 3-2
Injury 26 206
Giving up pig breeding 26 2-6
. Too fat or too big 2.1
Labor difficulties 200

21

20

Miscellaneous 0
Total 10%?

¥

The above survey was summarized by Pomeroy as follows:

(1) Sixty-four percent of the sow disposal was due

- 11 -

to "failure to breed", "piglet mortality", "old age" and "low
fertility".

(2) The average breeding life of 1000 sows was 3.75
litters per sow which agreed closely with earlier work of
Donald (191.1) who reported an average of 3 or u litters in
a lifetime. Pomeroy found only two litters per sow was the
modal value.

(3) In the sows which failed to breed (21.h per-
cent), the incidenoe of reproductive failure was greatest
in young females. "Of all the sows discarded as sterile,
30.3 percent were discarded after having had one litter.”

(h) The failure to conceive was highest in sows
which farrowed at an age of under twelve months.

(5) Sows which farrowed for the first time at 1h to
15 months had a longer breeding life time and produced more
pigs per litter and more pigs per sow's lifethme.

(6) Mortality of the baby pigs accounted for 17.8
percent of the sow culling. The average pro-weaning‘mor-
tality found in 2u11 litters was 26.5 percent. Pomeroy noted
an unexpected number of litters which had 100 percent mor-
tality of the pigs born. He believed death in.these cases
was due to "failure of adaption to a post-natal environment
because most of the pigs which die are undersized and weak-
ly at birth." He acknowledged the fact that there may be
cases where infection kills all pigs, weak ones as well as
the thrifty ones.

(7) Low fertility was responsible for culling 10

percent of the sows. In most cases, the animals culled were
young sows which produced small litters because of low ov-
ulation rate, or possessed normal ovaries coupled with ex-
cessive embryonic death.

(8) 01d sows were culled at a modal number of
litters of 8 suggesting further management reasons at this
time for culling. These reasons might include making room
for young sows as well as those reasons mentioned previous-
ly, that is, excessive piglet mortality and uneven or un-
thrifty litters.

Embryonic mortality versus infertility

Pomeroy (1960b) continued, after his initial survey,
with several experimental observations in regard to intra-
uterine physiology of swine and its effect on fetal develop-
ment.

In one experiment, sows were slaughtered in 2 groups,
the let at 7 days and the 2nd group at 12 to 21 days after
breeding. Subsequent to slaughter the reproductive tracts
were removed and examined.

0f the sows with normal ovaries, 7 out of 10 bred 7
days before slaughter were pregnant, but only ll out of 22
were pregnant when slaughtered between the 12th and 21st
day after breeding. This suggested to Pomeroy that the
main cause of sterility in sows, with apparently normal
ovaries, was embryonic mortality. 0n the other hand, of
sows with apparently abnormal ovaries at slaughter, only

one of 19 which had been mated to a fertile boar was

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pregnant. This suggested that of sows with abnormal ovaries
the main cause of infertility was lack of fertilization.
Fetal development
In another experiment, Pomeroy (1960c) compared data

obtained from an inbred herd of Large White pigs at the
Animal Research Station, Cambridge, with that from outbred
Essex pigs from swine farms of the area. The purpose of the
study was to find if growth and development of the fetus in
normal and inbred sows could be associated with neonatal
mortality. A summary of the experiment follows:

(1) Inbreeding increased preweaning mortality from
30 to 14.5 percent in the first four generations, from 50 to
68 percent in the 5th to the 9th generation and to 88 percent
in the 10th generation of inbreeding.

(2) During the first three days after birth 70.2
percent of all deaths occurred.

(3) The average birth weight of the baby pigs
which died the first three days was only 1003.5 grams com-
pared with 1258.5 grams for the surviving pigs. 0f the pigs
which died within three days, 83.0 percent weighed less than
900 grams at birth. 0f the pigs which weighed 1170.0 grams
or more, 18.5 percent died within the first three days.

((1) There was greater mortality during the winter.

(5) Mortality was highest in litters under 5 and
0V” 15. however those litters between 5 and 15 exhibited no
increase in mortality up to 15.

(6) High levels of inbreeding in Large Whites

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tended to slow fetal growth from mid-pregnancy. When the
inbred females were bred to boars of another breed the fetus-
es grew normally. Pomeroy (1960c) attempted to study this
problem further with reciprocal ova transplants between in-
bred.Large Whites and Essex, but was unsuccessful in his

_ attempt.

' (7) Both increased fetal age and increased litter
size affected fetal weight. Variation of fetal weight be-
tween litters could not be demonstrated statistically.

(8) Male fetuses were heavier than females at all
stages investigated.

(9) X-ray photographs revealed ossification was
mere advanced in the largest fetus within a litter when com»
pared to the smallest; however, the appearance of centers
of ossification were not delayed in the latter.

The growth curve of outbred fetal pigs from data of 80

outbred litters was found to be the cubic equation:

w =10 (0.21m? t - n.06)3
W = average weight of normal fetuses (gm.)
t = stage of pregnancy (days)
Pomeroy transformed the data into an equation used by
Huggett and Widdas (1951).
w1/3 s a(t - to)
W = fetal weight (gm.)

conception age (days)

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constant (specific fetal growth velocity)

to = constant

-15-

The formula for the growth curve was the equation:
w1/3 . 0.1135 (t-16.S9).

Pomeroy observed that the constant 0.1135 agreed favorably with
the figure of 0.12suggested by Huggett and Widdas (1951).

Pomeroy further found that there was a slight decrease
in average fetal weight with increased litter size only when
the litters past the 20th day of gestation were considered.
He contended that the effect of litter size on average fetal
weight was greatest after the placenta had reached its max-
imum development and competition began between fetuses.

Hammond (1960) made a similar contention. "In the
embryonic stage the embryo size is not affected by the plane
of nutrition of the mother for the trophoblastic cells have
priority of nutrition from the bloodstream over the maternal
tissues.” He then proposed that the area of the placenta
at the end of the embryonic stage determined what quantity
of nutrients the develOping animal would receive in the fetal
stage. The proposal was based on the theory that the" avail-
able nutrients during the fetal stage resulted primarily from
diffusion between the blood streams of the mother and the fetus.

In addition, Pomeroy found that the relationship between
litter size and within litter variation in fetus weight was
Significant and positive. Variability in fetus weight, there-
fore, increased with increased litter size. Pomeroy recalled
that Warwick (1928) had made a similar conclusion. Pomeroy
noted that variability in fetal weights increased after about
60 to 70 days which corresponded to the time when

 

-]_6-

the placenta had reached its maximum size. Hammond (Parkes,
1952) from experiments with rabbit litters of different
sizes, found after the thh day of pregnancy, that the fetus-
es began to vary in size. This time corresponded to the time
when the rabbit placenta had reached its maximum dimension.
Growth of fetal membranes

The membranes, according to Pomeroy (1960c), grew fast-
er than the fetus until 65 days and then stopped growing or
grew only slightly until near term. A second increase in
growth rate was noted at 100 days.

Growth of the fetal fluid

Pomeroy (1960c) confirmed Wislocki's 1935 research con-
cerning the growth of the fetal fluids. The allantoic fluid
reached its maximum volume at 65 days which indicated, accord—
ing to Pomeroy, an expansion of the uterus to make room for
the growing fetuses. Pomeroy found the increase in allan-
toic fluid volume was much less rapid in gilts than in sows,
and reached a maximum at 75 days compared with 65 days for
sows. The maximum was 150 grams of fluid for gilts and 1.00
grams for sows. There was a greater similarity between gilts
and sows in amniotic fluid volume. The amniotic fluid weight
was also less variable between fetuses than the allantoic
fluid weight.

Growth of uterus, cervix and vagina of sows

Pomeroy (1960c) found that the growth of the uterus was

rather complex. The following regression equations were

fitted to the data from the 30 to 90 day period post-breeding:

 

- 17 -

ll

(1) Sows Y 21.h8 x1 + 57.59 x2 + 28.03
(2) Gilts Y = 21.82 x1 + 36.87 x2 + 15.30

weight of uterus (grams)

Where Y
x1 = stage of pregnancy (days)
x2 = number of fetuses
This equation suggested that the stage of pregnancy had
a.similar effect on uterine weight in gilts and sows while
the effect of number of fetuses on uterine weight was greater
in.sows than gilts. Pomeroy said, "The increased growth of ,
the uterus with increasing litter size suggests that over-
crowding is not primarily responsible for decreased average
weight of fetuses in large litters."
Growth of the cervix and vagina in this study was not
significant until the 90th day in gestation. At that thme
a slight growth in length and appreciable growth in weight

and a softening and relaxing of the walls occurred.

Differential growth of the carcass
Pomeroy determined the percentage composition of fetal
carcass based on dissection into fetal joints. The dissec-
tions were performed according to the method of McMeeken
(1980) with minor modifications.
The growth pattern of the anatomical joints indicated
a decrease in the percent of head, neck and pelvis: the per-

cent loin and leg increased and the thorax and shoulders as
a percentage of total weight tended to remain constant.
The carcass weight of the fetus as a percent of the

total fetal body weight increased in the case of the Large

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Whites from 73.9 percent at 51 days to 81.8 percent at 97
days and then fell to 78 percent at 110 days. In another
group of Large Whites, the value at 51 days was highest (80.14.
percent) and then decreased to 76.6 percent at 110 days. The
trend continued until birth of the baby pigs. At birth the car-
cass weight was approximately 75 percent of the body weight.
Growth of the skeleton
The fetuses were divided into skeleton and flesh by
dissection. The total skeleton was expressed as a percent
of the total carcass weight. The weight of the parts of the
skeleton were expressed as percentages of the whole skeleton.
The results, as found by Pomeroy, are briefly stated
as follows:

(1) LargeWhites had a higher percent of skeleton
than Essex. At 110 days (near term) the Large White averaged
25.0 percent vs. 21.9 percent for the Essex. In both breeds
this value fell between 51 and 714. days and rose again at 97
days. After 97 days, the percent rose slowly for the Large
White and remained almost constant in the Essex.

(2) Weights of the bones of the head, as a percent
of total skeleton, fell between 51 days and 7).; days then rose
aBasin at 97 days with a slight rise to term.

(3) Weight of the cervical vertebrae, as a percent
of the total skeleton, remained fairly constant (11.5 to 5.5
Percent) from 51 days to term.

(1;) Weight of the thoracic skeleton, as a percent
0f the total skeleton, decreased from 51 to 714. days and then

-19-

increased to 97 days, remaining fairly constant until term.

(5) Weights of the lumbar vertebrae, as a percent
of the total skeleton, increased between 51 and 71;, days then
decreased between 714. and 97 days and thereafter remained
fairly constant.

(6) Weight of the pelvic skeleton, as a percent of
the total skeleton, decreased between 51 days to 7).; days
and remained level after that.

(7) Weight of the skeletal bones of the legs, as
a percent of the total skeleton, increased steadily to term.

Pomeroy suggested that the data indicated the following
pattern: "The weight of the skeleton increased less rapidly
than the carcass as a whole, so that it became a progress-
ively smaller proportion of it. In so far as the percentage
of bone in the Essex fetus was lower than in the Large White
there is a suggestion that it had reached a more advanced
state of maturity."
Comparison of the largest and smallest fetuses
within litters
The develOpment anatomically and chemically of fetal

pigs was used by Pomeroy (1960c) as a criterion to test the
hypothesis that subnormal birth weight could be used as an
index of prematurity. It was his contention that a pig which
is less than average weight at birth exhibits characteristics
of an earlier fetal age. He compared the smallest with the
largest at four periods; 7).; days, 91+ days, 1014. days and 110
days.

-20-

Comparisons were made of weights of several measures
of the smallest fetus expressed as a percent of the weight
of a similar measure of the largest fetus. These ratios of
body weight, carcass weight and of several weights of internal
organs revealed that the differences between the smallest and
the largest diminished toward birth. It was noted that the
carcass measurement was more affected than weight of organs,
particularly the brain weight which was hardly affected at
all.

When the skeletal weight and carcass weight of the
smallest fetus of a litter were compared to the largest, the
ratio of the weight of the skeleton of the smallest to that
of the largest fetus was consistently larger than the ratio
of carcass weights except at 110 days, when Pomeroy reported
a small difference between the smallest and the largest fetus.

Also the skeleton as a percentage of the carcass
weight of the largest fetus indicated a similar pattern in
that the skeleton of the larger fetus was generally a smaller
part of its carcass than was the skeleton‘of the smaller
fetus as a part of its carcass. From this data Pomeroy said,
"----the slower growth of the smallest fetus affects the
growth of the skeleton less than the carcass as a whole or,
in other words, the growth of the musculature of the carcass
was retarded to a greater extent than the skeleton."

As the weight of the total skeleton of the smallest fetus
expressed as a percentage of the weight of the skeleton of

the largest fetus was increasing during pregnancy the

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differential growth of certain skeletal parts was following,
in general, a similar pattern. Expressed as a percent of the
total weight of the skeleton, the weight of the legs, shoulders,
pelvis and loin increased during the period of 7h days until
term. The relative weight of the thorax, neck and head re-
mained steady the last three weeks of gestation but decreas-
ed slightly during the three week period from 7h to 97 days.
In general these differential measurements revealed no great
difference between the largest and the smallest fetal pigs
when the two sizes were compared at any of the four periods
studied, i.e. 7h. 97, 108 or 110 days.

Comparisons of the chemical composition of the smallest
and largest fetuses were also accomplished. Pomeroy cited ‘
the work of Mitchell g£_al. (1931) who determined percent-
ages of dry matter, calcium, phosphorus and nitrogen within
the fetus. Mitchell's work is reviewed later in this review
of literature. Pomeroy also cited work of McCance and Wid-
dowson (195h) who had described changes in sodium, potassium
and thloride concentration in the fluids of the prenatal and
postnatal rat. Newland (1955) and Newland and Davis (1961)
also presented data of fetal uptake of manganese and phos-
phorus.

Pomeroy (1960c) determined the concentration of water,
total nitrogen, protein, Cu, Mg, P, Na and K in the fetus
from 51 days to birth. The findings were:

(1) Percent of water decreased

(2) Total N percent increased
(3) Protein percent increased

 

- 22 -

Ca increased markedly (5 times)
Mg increased markedly (2 times)
P increased markedly (3 times)
Na increased slightly
K increased slightly

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He found the concentration of potassium somewhat lower
in inbred Large Whites than in outbred Essex. Spray and
Widdowsen (1951) earlier had determined sodium and potassium
content of pigs at birth. They found the concentration of
sodium fell and the concentration of potassium rose after
birth until a constant was reached. Pomeroy noted the inbred
Large Whites were lower in potassium than the Essex in all
stages indicating perhaps that the inbred pigs were more im-
mature. There also was a general tendency for the concentra-
tion of sodium to fall and potassium to rise. The overall
picture was clouded somewhat by a sharp rise in percent sod-
ium and a fall in percent potassium at 108 days to 113 days.

These chemical determinations were applied by Pomeroy
to test the hypothesis that smaller fetuses were more im-
mature. He reported that the percent water was lower in the
larger fetuses at 108 days and 113 days and the percent of
nitrogen was higher in the larger fetuses at all stages. In
general, the differences in each case were small and the
hypothesis, according to Pomeroy, could not be supported.

Ossification of the skeleton: Pomeroy (19600) noted
that large fetuses not only had larger bones but the fetuses
were more advanced in degree of ossification. However, the
time of appearance of centers of ossification was not re-

tarded in the smaller fetuses. In early studies of fetal

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growth in the rabbit, Appleton (1929) found heavier fetuses
possessed more ossification. Wallace (19348) found greater
ossification in fetal lambs from ewes on a high plane of
nutrition.

Prenatal mortality

Pomeroy (l960d) approached his study of prenatal mortal-
ity by using three different measures. These were (1) loss
of ova by comparing the number of corpora lutea with the
number of fetuses, (2) percent of degenerate fetuses of the
total number of fetuses and ( 3) relationship between ova
shed and the loss of ova.

The overall percentage loss of ova expressed, as a
percentage of corpora lutea count, was 38.914. Percent. Most
of this loss had occurred before the end of the 20th day.
The loss after 20 (days was determined at 10 day intervals.
There was no statistical difference noted between any 10
day period from 20 to 89 days; however, the loss between 90
to 113 days was highly significantly different and greater
than between 20 and 89 days past breeding. Pomeroy noted
his data agreed with Warwick (1928) who indicated that as
Pregnancy proceeds fetal losses increase.

His second measure was the percentage of degenerate
fetuses as compared to the number of fetuses. These data
did not show that time and prenatal mortality were related.
One reason proposed by Pomeroy was the difficulty of observ-
ing fetuses which were undergoing autolysis early in preg-
nancy. The following table from Pomeroy (l960d) indicates

- 2h -

that the percentage of degenerate fetuses reached a peak at

60 to 69 days.

Table 2. Percent degenerate fetusesl (Pomeroy, l960d)

 

Stages of pregnancy Fetuses, percent degenerate
da a);

20-29 0.00
0-g9 9.21
O- 9 h-Z9

50-59 h-17

60-69 17.71

70-79 7e8

80-89 8e8

90-99 Seq?

100-113 1.26

Tatal 7e67

The third criterion of Pomeroy was the effect of the
number of ova shed on the loss of ova. The data from gilts
killed during the first 10 days after conception, suggested,
”in cases of normal ovulation rates, i.e. up to about twenty,
it is rare for only some of the ovum to be fertilized, i.e.
all the ova are fertilized or none are fertilized." He noted
that, ”there were no‘significant differences in percent mor-
tality with increased ovulation rate between 11 and 22, but
there was a significantly greater loss in litters where the
ovulation rate was 23-25." This was an exceedingly interest-

198 observation which needs further experimentation to sub-

stantiate .

I The table is a portion of Table 11, Pomeroy (l960d).

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2. Fetal nutrient deposition as a measure of food requirement
of pregnancy in swine.

Mitchell M. (1931), in their classical study of the
food requirements of pregnancy in swine, noted the import-
ance of adequate nutrition during late pregnancy. They found
that two-thirds of the growth of the fetus occurred during
the last four weeks of gestation. Gross energy, crude pro-
tein, ash, calcium, phosphorus and iron all progressively
increased in rate of depostion during pregnancy. At 16
weeks (near term) the deposition was double the amount at
10 weeks.

Sixteen pregnant gilts were sacrificed starting at five
weeks post-breeding to obtain an estimate of the energy need-
ed for gestation. A detailed summary of their observations
are listed below:

(1) Gilts in metabolism cages required five pounds
of feed per day. Group fed pigs required eight pounds. Both
groups were fed to gain 1 to 1.5 pounds per head per day.

(2) After 16 weeks of gestation (112 days), the
uterus accounted for 29.8 percent (6 fetuses), 28.1 percent
(7 fetuses), or 36.0 percent (10 fetuses) of the live weight
gained during pregnancy.

(3) Variability in fetal weights within litters
measured by coefficient of variation, decreased from 18.8
percent at 35 days to 3.9 percent at 63 days, then increased
to 20 percent at 90 days and remained above 10 percent until

98 days and was irregular from 111 days until term.

 

 

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(li) Male fetuses were heavier than females by 5.5
It 1.11 percent after the sixth week.

(5) Dry matter increased regularly from 8.15 to
8.90 percent at 35 days to a range of 16.81 to 17.53 percent
at 105 to 112 days.

(6) Ash content of the fetuses on a dry matter basis
increased from 12 to 22 percent from the 5th to 11th week
with no further increase during the last five weeks.

(7) The calcium concentration of the dry matter in-
creased irregularly up to termination of pregnancy as did the
calcium concentration of the ash.

(8) The phosphorus concentration of the dry matter
increased irregularly up to term, but its concentration in-
creased even more irregularly than the calcium percent of the
ash. _

(9) The percentage of iron en a dry matter basis
did not show any progressive variation. Liver and spleen
iron concentration equaled or was greater than that in the
remainder of the fetus near term, which indicated storage of
iron.

(10) Fat did not show a progressive increase.

(11) Intrauterine nutrient deposition was determin-

ed by the equation:
w = kt“

W equals weight of the nutrient deposited in the uterus at
the end of each gestation week t. W was expressed in grams

except for iron which was in milligrams and gross energy which

 

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was in calories. The symbols k and n were constants to be
determined from each set of data. Constants k and n were
determined by the method of least squares for total fresh
weight of products of conception, total gross energy, total
crude protein, total ash, calcium, phosphorus and iron. All
the data were corrected to a litter size of eight. The e-
quation mich was used to express fetal growth was also used
by Murray (1925), MacDowell and Allen (1927) and Mitchell
(1929).

(12) Using the first derivativepof the above
equations a deposition of 312 grams of new material was pre-
dicted at the 16th week of gestation (just prior to birth)
for a litter size of 8, of which 33 grams would be crude pro:-
tein, 11.7 grams ash, 11.29 grams calcium, 1.98 grams phos-
phorus and 12.3 milligrams iron. The caloric content of
this material was 272 calories. At the 10th week nutrient
deposition rates would be only one-half or less of the rates
at 16 weeks.

A more recent paper using similar criteria was published
by DeVilliers M. (1958). Nine gilts, one each at 10 day
intervals, from 30 to 110 days post-breeding were used. The
following measurements of uterine nutrient deposition were
1“Ported: organic dry matter, nitrogen, crude fat, ash, cal-
cium, phosphorus and iron. These measurements were then used
to calculate the energy content of the uterus and to prepare
feeding standards. A close relationship was found between

the size of fetus and the time from conception. Based on

 

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their data and Mitchell's 1931 experiment, the following ex-
ponential equation was developed for the deposition of total
nitrogen.
n (grams) = 9.8 .0-031t

Log N (grams) 8 0.0135t + 0.990

t 8 days after conception

These workers found that nutrient deposition increased as an
exponential functionof time from conception amich means
"considerable deposition first takes place during the latter
part of gestation.“

Similar equations were formulated for total deposition
of energy, iron, calcium and phosphorus in the products of
conception corrected to a litter size of ten. They also pre-
sented equations of daily deposition of nitrogen, energy,
calcium, phosphorus and iron corrected to a litter size of
ten.

0n the basis of daily nutrient deposition and by con-
sidering the utilization of ingested nutrients and the heat
increments of gestation, nutritive requirements for fetal
developuent were calculated and feeding standards were es-

tablished for the pregnant female.

- 29 -
3. Development of intestinal carbohydrases and acid and
alkaline phosphatase in the pig.

anon of the early research regarding the fetal develop-
ment of digestive enzymes of mammals was reviewed by Needham
(1931). Later research was reviewed by Driscoll and Hsia
(1958) and Fries (1958). Hartman gt_gl. (1961) surveyed the
literature regarding the digestive enzymes of the neonatal
pug and the pig through weaning. Driscoll and Hsia's review
highlighted the research as applied to human pediatrics,
while Fries reviewed enzymal studies of domestic animals,
particularly the calf. Fries also reviewed the formulation
(and‘utilization of milk replacers for calves, utilization
of various carbohydrates and protein sources by calves and
baby pigs and the effects of enzyme supplementation of prac-
tical calf and baby pig rations.

The following survey of the literature will briefly re-
view the development of the digestive carbohydrases and in-
testinal phosphatases of the pig. A detailed discussion of
the other gastro-intestinal enzymes can be found in the re-
views above.

The survey is divided into two main parts which are
as follows: (1) neonatal and fetal development of the carbo-
hydrases of the pig and (2) development of intestinal phospha-
tases.

Egoqatgl and fetal developmentfigf the intestinal carbo:

hydrases of the pig

The following section reviews (1) utilization of carbo-
hydrates by the neonatal pig and (2) carbohydrase activity

 

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of the fetal pig and neonatal pig.

Utilization of carbohydrates by the neonatal pig

Johnson (l9h9) found glucose or lactose fed in purified
diets, beginning at the 2nd day after birth, produced satis-
fhctory growth in pigs. When.sucrose was fed, acute diar-
rhea resulted which led to kidney hemorrhage and death with-
in.a ahort time. Fructose was also found to be unsatisfactory.
The inability of the two-day old pig to utilize sucrose had
been confirmed by Becker gg_gl. (l95hb). Becker and his
associates also found glucose or invert sugar produced satis-
fictory growth and survival rates of pigs frmm 0 to 9 days.
Pigs that received fructose or sucrose lost weight and had
a very low survival rate. Diarrhea was severe with the use
of fructose and sucrose, less severe with invert sugar and
there was none with glucose.

: Two studies'have been reported in which various carbo-

hydrates were compared for the pig in the period from 7 to
35 days of age. Becker gt_gl, (l95ha) found that glucose,
lactose, sucrose, dextrin or starch produced about equal gains
during this period. Less diarrhea was noted in the groups
receiving lactose and starch. Hudman gg_§;. (1955) reported
that lactose was superior to glucose, sucrose, corn syrup
solids and corn. Corn starch, oat greats, corn flakes and
gelatinized starch produced gains considerably lower than
lactose.

Using 9 week old pigs Becker and Terrill (l95h) found

that glucose, sucrose, dextrin and starch produced equal

 

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gains when fed at the rate of 50 percent of the diet. A
slight depression in growth occurred when lactose was fed at
this rate, but the depression did not occur when the diet was
composed of 25 percent lactose.

Cunningham and Brisson (l957a,b) using two-day old baby
pigs observed the effects of supplementing various purified
starch-containing diets with amylolytic enzymes. Supple-
mental pancreatic and malt amylases had no effect on growth
rate, survival time, or digestibility of raw or cooked starch.
Cooked starch was inferior to raw starch as a source of car-
bohydrate in baby pig diets.

Cunningham and Brisson (1957c) also studied the utili-
zation of maltose by newborn pigs. Through the use of diges-
tion trials and intestinal 100p techniques, they found orally
ingested maltose was 97.14 percent digested by 2 to 5 day old
pigs. The rate of digestion was 0.72 grams per kilogram of
body weight per hour. Maltose, injected into a tied-off
segment of the small intestine of newborn to L; day old pigs
was digested at the rate of 0.62 grams per kilogram of body
weight per hour.

By following the level of the reducing sugars in the
blood Dollar and Porter (1957) and Dollar _e_’§___a_]_.. (1957) dem-
onstrated that the young calf and pig cannot utilize maltose,
dextrin or starch for the first )4 to 5 weeks of life and
sucrose for at least 7 weeks.

Braude 23413;. (1958a,b) found the beginning of maltose
utilization by the pig came at 5 days of age and at that

 

 

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-32-

time the performance was similar to that of glucose when glu-
cose was fed. They also discovered that the S to 7 day old
pig could begin to utilize solutions of dextrin. Therefore,
they concluded that amylase was secreted in effective amounts
by pigs of this age. They found that rapid digestion of these
food stuffs was important for their assimilation and the pro-
cess depended on the age of the pig and the solubility of the
polysaccharide. They suggested it was possible, therefore,
that the action of amylase was complemented by a solubiliz-
ing process that was inadequate in the young pig but that
developed with age.

Carbohydrase activity of the fetal pig

and neonatal pig ‘

Lactase activity: Plimmer (1906) came to the conclusion
that lactase was present in adult omnivores and carnivores
but not in adult herbivores. Mendel (1906) was notable to
demonstrate lactase activity in the adult pig, an omnivore.
It was interesting to note that assays of swine intestinal
tissues for lactase were not performed again until the 1950's
when Bailey M. (1956) found lactase to be present at a
.high level at birth in the pig. These Canadian workers
measured the lactase activity from birth to 50 days of age.
The activity was high until 20 days then decreased steadily
to a negligible level at 50 days. Heilskov (1951) also
found a similar pattern for rabbits and cattle.

Lactase activity was studied by Walker (195%) in the
Pig from birth to 5 weeks. When the activity was expressed

 

 

-33-

per kilogram of body weight per gram of dry tissue, the acti-
vity declined rapidly in agreement with the data of Bailey
M. (1956). When the activity was expressed as total lac-
tose hydrolyzed per hour per animal, the amount of lactase
present at birth remained at a constant level up to 5 weeks
of age. walker suggested that this indicated a fixed amount
of lactase producing tissue was present at birth. Walker
said, "It is merely shared out over the length of the intes-
tine as the pig grows." This method of expressing the act-
ivity of lactase corrected for the growth of the intestinal
tract. He believed it gave a more correct picture of the
total amount of enzyme activity present.

Fries (1958) in his review of the development of lactase
in human fetuses reported that lactase is rather late in its
appearance. It was not reported until the 7th or 8th month
and often times was absent in prematures. Mendel (1906) was
able to find lactase in the pig embryo, but he did not re-
port the age of the embryos.

Pancreatic amylase activity: Cunningham (1959) stated
his belief that more information about digestion of starch
by the neonatal pig is necessary to decide whether it is
possible to improve the pig's utilization of starch.

Kitts M. (1956) in a quantitative study using pigs
from birth to 37 days found pancreatic amylase activity was
approximately 100 units per kilogram of body weight at birth,
increased to 1500 units at the 6th day and remained at that
level through the 22nd day of age. It had increased to

 

 

-3u-

15,000 units at 37 days. Hudman M. (1957) and Walker
(1959b) also found a similar pattern for this enzyme.

Reports of the occurrence of pancreatic amylase ‘in the
fetus appeared to be in conflict according to Fries (1958)
and Needham (1931). The presence of pancreatic mlase had
been both reported and denied between the 5th and 6th fetal
month in the human. It also had been reported as present
and absent in the newborn and the one month old human infant.

There were few experiments concerned with amylase in
other species. Langendorf (1879) found pancreatic amylase
to be present in the 100 millimeter pig embryo and the 250
millimeter bovine embryo. He found the rat fetus and the
newborn rat possessed amylase activity, but this activity was
absent in the newborn rabbit.

Haltase activity: The importance of the enzyme maltase
in swine nutrition is based on the premise that large amounts
of amylase are present soon after birth, whereby large amounts
of maltose and dextrin are formed from starch. Although in
some cases it is believed that maltose can be absorbed intact
from the intestine, it is generally accepted that maltose
must first be hydrolyzed to glucose (Cunningham, 1959).
Therefore, it is self evident that maltase is necessary for
maximum utilization of the economically important starchy
foods by the post natal pig. 6

As early as 1906, Mendel reported maltase in the gastro-
intestinal tract of the) suckling pig. Bailey M. (1956)

reported intestinal maltase activity was very low in the new-

-35-

born pig but increased rapidly the first 25 days and romaine
ed at that level for 50 days. Cunningham.and Brisson (1957b,
(fl, using the ligated intestinal loop technique, found maltose
disappeared at a rate of 0.66 grams per kilogram of body
weight per hour and was 97.14. percent digested by 2 to 5 day
old pigs. ‘

Cunningham (1959), using several techniques including
digestion trials and carbohydrate tolerance curves, found
‘maltose was 95.h percent digestible and the rate of digestion
of maltose did not approach the rate of digestion of glucose
until the pigs were 7 days old.

Walker (1959b) found maltase in the pancreas increased
from low levels at birth to high levels at 5 weeks. Small
intestinal maltase, expressed as maltose hydrolyzed per hour
per animal, also increased as age progressed. It was pre-
viously stated that lactase did not progressively increase
when expressed on an animal basis. Walker noted anemia in
pigs tended to delay the normal development of maltase pro-
ducing tissue and the increase in.maltase activity was de-
layed to’a later age. Walker suggested that although there
appeared to be sufficient amylase at all stages, maltase ap-
peared to be limiting in.the early life of the pig.

In the swine fetus, maltase was found to be active very
early in fetal development by Mendel (1906): however, he did
not indicate the age of the fetus.

Sucrase activity: As noted previously, Becker g£_al.

(195hA,b) and Becker and Terrill (1958) found the neonatal

 

. ,av

...~-
.

”.II‘

In!

03/

cg..-
.a

doe

. I
In

‘h‘

' I
(-
4‘,

 

-36-

pig was unable to utilize sucrose until one week old. These
experiments were based on digestibility of the sugars and
growth data of the pigs.

Enzymatic sucrase assays were performed by Bailey gt_gl.
(1956) and Walker (1959b). Sucrase activity was found to be
very low from 0 to 10 days and increased to a high level at
5 weeks of age. Mendel (1906) could not detect sucrase in
the fetuses of swine.

Gastric acidity: Although not a digestive enzyme, the
development of gastric acidity is discussed because of its
effect on the reactivity of digestive enzymes. Kvasnitskii
and Bakeeva (l9h0) found that hydrochloric acid did not ap-
pear in significant quantities in the pig until 20 to 30
days of age. Lewis g§_gl. (1955) recorded pH values of n.3,
3.5. 3.9, 3.6, h.0 and h.3 on six-hour-fasted baby pigs stom-
' achs at l, 7, 1h, 21, 28 and 35 days of age, respectively.
Walker (1959a) followed the formation of NaCl and total acid-
ity of the stomach contents of Large White pigs from birth
to 5 weeks of age. The average pH of the duodenum.and small
intestine was 6.5 and 6.u respectively. He verified that
total NaCl and total.acidity in the stomach remained nearly
constant from birth to 5 weeks. No free HCl was detected at
any age.

Grutzner in 1875 (Fries, 1958) was unable to find acid
in.the fetal stomachs of cattle, sheep, pigs and dogs. The
stage of fetal development was not reported. Moriggia in

1876 (Fries, 1958) reported that the stomach contents of the

-37..

bovine fetus were always slightly acid after the 3rd month.
Hydrochloric acid was found to be present in the human fetal
stomabh as early as the 19th week of development (Keen and
Hewer, 1929).

Stomach contents of premature infants were often report-
ed to be lower in acid content than the contents of full term
infants (Miller, 19141). In general, the acid content was
relatively high at birth. It dropped to a low at 10 days of
age and then increased.

A pH of 14.1; for the combined contents of the omasum and
abomasum of calves at birth was reported by Parrish and
Fountain (1952). For the rumen and reticulum, a pH of 6.);
was recorded. Since the omasum does not secrete hydrochloric
acid, a somewhat lower pH would be expected if the abomasum
had been considered alone.

Occurrence of intestinal alkaline and acid phosphatase

Florence Moog of the Department of Zoology, Washington
University, St. Louis, Missouri became interested in the
presence and maturity of certain families of enzymes which
catalyze the hydrolysis of a single phosphate group. These
enzymes are called phosphatases, more correctly phosphomono-
esterases. According to Fruton and Simmonds (1958), phospho-
monoesterases have been studied extensively, and a number of
distinctly different enzymes of this group are known. The
best characterized are those in blood plasma, milk, intesti-
nal mucosa and bone. A 133 of 9 is optimum for activity of

the alkaline variety and a pH of 5 is optimum for the acid

 

 

.
fl
'1’ ‘-
rr‘"
.

:.:-‘

'0

a
l~o
is

[7"

a)

be
.

c"?

k..-

- 3a -

phosphatase. The alkaline phosphatase is further character-
ized'by need for divalent magnesium ions for maximum acti-
vity. It is also known that phosphates inhibit its action.

In l9uh, Moog published the first of several papers discussing
the pattern of differentiation of phosphatases of the in-
testinal tract.

Moog's initial paper in l9hh entitled, “The localization
of alkaline and acid phosphatase in the early embryonogenesis
of the chick," noted that the phosphatase enzymes accumulated
in specific areas including the intestine. This initial
study was followed by several papers from.l950 to 1958 all
associated with these enzymes and related phenomena.

The evidence presented by the group at St. Louis sug-
gested the following conclusions:

(1) The production of intestinal alkaline phos-
phatase followed a definite time pattern during development
of'the chick and mouse (Moog, l9uh). The enzyme system was
found in the epithelial cells of the intestinal mucosa. In
the chick embryo, its level of activity rose 1000 times dur-
ing the lasttwo and one half days of embryonic life (Moog,
1950). In the mouse, a rise in activity was seen just be-
fore birth and just before weaning (Moog, 1951 and Moog,
1953). In the guinea pig the level was slightly elevated in
the young animal but soon fell back to the level of: the new-
born which then persisted during adulthood (Moog and 0rtiz,)
1957).

(2) "Physiologically active" glycogen concentration

 

‘n
(to

7"

I.

V‘

 

 

-39..

(Bloom 9.2.52.0! 1951) increased in the intestinal epithelial
cells at the same time that the alkaline phosphatase increas-
ed (Moog and Wenger, 1952; Moog and Richardson, 1955; Moog
and Thomas, 1957).

( 3) Acid phosphatase concentration did not increase
as markedly as alkaline phosphatase during the fetal period
but tended to remain constant (Moog, 19146; Rudnick, 1958).

(h) Adrenal glucocorticoids produced endogenously
or injected from an exogenous source produced an earlier ap-
pearance of alkaline phosphatase and glycogen. The early
increase in alkaline phosphatase equaled the normal maximum
of the controls, while glycogen surpassed the normal level
of the controls (Verne and Hebert, 19149; Moog and Richardson,
19553 Moog and Thomas, 1957; Moog and Ford, 1957). These
data hold true for the chick, the mouse and the rat.

(5) Desoxycorticosterone glucoside produced a weak
effect in the stimulation of the earlier appearance of alka-
line phosphatase and glycogen as discussed above. This sug-
gested that mineralocorticoids may not have a function in
the mechanism in the same way that glucocorticoids stimulated
the earlier appearance of alkaline phOSphatase and glycogen
(Moog and Richardson, 1955).

(6) During the time when the epithelial cells were
increasing in their phosphatase content, these cells were
also making dramatic changes in microscOpic appearance. Under
the light microscope they changed from cuboidal and became

columnar, with slender elliptical nuclei and with dense

-uo-

cytoplasmic layers. The cytoplasmic layers appeared first

above the nuclei and then basal to the nuclei. The striated
borders became composed of neutral polysaccharide on the sur-
face of the lumen. The cytoplasm first became more and then
less basophilic and the accumulation of glycogen was transient
(Moog'and Richardson, 1955; Rudnick, 1958).

(7) Addition of sodium phenyl phosphate substrate
after the 16th day to the chick embryo increased the level
of'the alkaline phosphatase but decreased the level of acid
phosphatase. This phenomenon occurred also when tissues
were cultured in vitro (Kato and Moog, 1958).

(8) Chick embryos injected with adrenal gluco-
corticoids were not retarded in growth if the injection was
made after the 16th day; the embryos were slightly heavier
than the controls, with heavier yolk sacs, which were drawn
in a little earlier before hatching than in the controls
(Moog and Richardson, 1955).

(9) The alkaline phosphatase was associated with
(the striated border of the epithelium (Zetterqvist, 1956;
Brandes gt_§l,, 1956; Puchtler and Leblond, 1958), and pos-
sibly was functional in transport of nutrients through the
epithelial cell into the blood.

MATERIALS AND METHODS

Experimental subjects

The fetal and newborn pigs were obtained from.l9 York-—
shire first-litter gilts. Supporting information was gather-
ed from h Duroc second-litter sows and 7 Hampshire x Duroc
gilts. The 19 Yerkahire gilts were selected for uniformity
of size and age from.the Michigan State University swine herd.
The Duroc gilts were positive control animals from a vitamin
A study. The Hampshire x Duroc gilts were from a study where
a.progestational hormone was adminstered pg; 25 to control
estrus. ~The females were all considered normal.

The 19 Yorkshire gilts were divided into 5 groups, bas-
ed on 21 day intervals starting at the 13th day post-breeding.
The 5 periods with the number of gilts and fetuses at each

period are listed below:

Yorkshire 30 days 3 litters 32 fetuses
51 days 5 litters 5h fetuses
72 days A litters 59 fetuses
93 days h litters 51 fetuses
Term 3 litters 37 Pigs
Durocs Term. u litters ks pigs
Hampshire x hS days 7 litters 63 fetuses
Durocs
Feedigg

The Ybrkshire gilts were self-fed ration 1, Table 3a.
The Duroc sows were hand-fed ration 2, Table 3b, and the
Hampshire x Duroc gilts were hand fed according to Nellor

2t 81. (1961). The rations, in.all.cases, were considered

-m-

-142-

Table 3a. Ration.l - self-fed to Yorkshire gilts

 

 

Ingredient Percent
Corn 67.8
Oats 10.0
Alfalfa meal 10.0
Soybean meal(uu%) 11.5
Meat and bone scrap 3.5
Limestone 0.h

Super trace mineral salt 0 5
Dicalcium.phosphate + zinc 0 2
VitaminB-supplementa 1.0 lb./ton
Vitamin A and D mixb 0 5 1
B12 supplement° l 0

9'2 grams riboflavin, u grams pantothenic acid, 9 grams nia-
bcin and 10 grams choline per pound of supplement
b(4.,lt50, 980 I. U. Vitamin.A and 1,26h,07h I. U. Vitamin D
per pound
°9 milligrams per pound of supplement

Table 3b. Ration 2 - hand-fed to Duroc sows

 

 

 

 

Ingredient Percent
Oats 7‘ w 3 . *7
Wheat 5 0

Soybean.meal (hh%)

Meat and bone scrap

Dried corn distillers solubles
Trace mdneral salt

Limestone

O O

O

#Hmooppmop
oommmooory

Vitamin B supplementa . 1b./ton
812 supplementb . 1b./ton
'Vitamin D supplement9 .5 gm./ton

Vitamin A supplementd

 

5&2 grams riboflavin:_h grams pantothenic acid, 9 grams

b niacin and 10 grams.choline per pound of supplement
9 milligrams per pound of supplement

° 1h2,000 I. U. per gram

d.l6 milligrams per kilogram body weight daily

- “3 -

nutritionally adequate .

Breeding
At the appropriate estrous periods, the Yorkshire gilts

were bred to 2 Yorkshire boars, one boar serving a gilt once
on the 1st day followed by a 2nd boar on the succeeding day.
Three of the Duroc sows were bred to Yorkshire boars and the
hth to a Hampshire boar and were also bred on 2 consecutive
days. The HMpshire x Duroc gilts were bred to Yorkshire
bears on 2 consecutive days.

Surgical procedure

The gilts which supplied the fetal pigs were taken from
the swine farm to the Michigan State University Veterinary
Hospital after being off feed and water for 21; hours. At the
hospital, caesarian sections were performed by the staff
veterinarians. A state of unconsciousness was produced in
the gilts prior to surgery by intravenous administration of
thiamylal sodium (Surital sodiuml) into a marginal auricular
vein. Anaesthesia was (maintained by further administration
of this compound or by (ether inhalation.

Manipulation of the fetuses and newborn:pig_s_

Hemostats were used to prevent loss of blood from the
fetal umbilical cord prior to weighing. The fetuses and new-
born pigs were killed by exsanguination prior to dissection.
Table )1 lists the anatomical measurements performed. Body

weight, crown-rump length and head width were measured.

E

1 Park Davis and 00., Detroit, Michigan

- up -

immediately after removal from the uterus or just following

births

The weight of the internal organs and length of the

dissected humerus was obtained immediately subsequent to

dissection. The other length measurements were from.X-rays

and involved the calcified diaphyses of the listed bones.

Table h.

Measurements of fetal growth

 

 

Weight of-

 

Body L. Gonad

hiver R. Gonad

Spleen Thyroid

L. Kidney Heart

R. Kidney Lung

L. Adrenal Brain

R. Adrenal Pituitary
Width of-

Head

3 Crown-rump length.

length of-

Body (c-r)a
Humerus (h-c )b
Humerus (t-c ) °
Humerugd

Radigs

Ulna

Metacarpal (3 or u)d
Metacarpal (2 or 5)d

b Length, head to condyle.
° Length, tuberosity to condyle.
d Calcified diaphyses length from.X-ray photographs.

Tissues for enzyme analysig

Iliumfi

Ischiamd

Femur

Tibiad

Fibulad

Calcaneousd
Metatarsal (3 or h)d
Metatarsal (2 or 5)d

Three fetuses from each of the Yorkshire and Duroc lit-

ters were chosen for enzyme analysis.

3rd and 5th fetuses in the right uterine horn were used.

In general the lat,

The

fetuses were opened by a ventral incision exposing the vis-

cerae

The liver and spleen were removed and weighed.

The gastrointestinal tract was removed and divided into

stomach, cranial duodenum, caudal duodenum, 3 portions of

-15..

the jejunumeileum, colon and rectum. The pancreas was also
ruoved. Imediately after dissection, the saniples were
placed in a glass vial, corked tightly and frozen in dry ice.

A small portion of each section was fixed in FAA1 for
histological examination.

X-rays of the fetuses

The subjects were placed on their left side in a natural
position directly over X-ray film. The film was protected‘
from light by a paper envelope. Paper towels were placed
between the film.packet and the fetus to absorb excessive
moisture. By placing the animal directly over the film, a
nearly identical image of the ossified area of the fetus was
produced on the film.by the X-irradiation. The amount of
irradiation was varied for each litter depending on the
thickness of the fetuses.

Length measurements were made of the ossified diaphyses
of the long bones.

Organ dissection and organ weights

The internal organs were removed from.the animals, dis-
sected free of connective tissue and weighed immediately.
The balance used was either a Roller-Smith double pan bal-
ance or a Mettler Model B, single pan balance. The choice
of balances allowed immediate weighing and the recorded value
for each organ was considered a fresh wet weight with minor

evaporative losses.

1 80 percent ethanol (95 percent), 15 percent formalin
and 5 percent acetic acid.

-k6-

After the skull cap and meninges were removed from the
brain, the spinal cord was severed at the atlanto-occipital
articulation and the brain was removed leaving the pituitary
in situ.

The pituitary was removed with a probe and a Sharp scal-
pel. This gland had definite form early in fetal life, there-
fore no problems were encountered in its dissection even
though it was quite small.

The thyroid was removed by blunt dissection and freed
of connective tissue under a dissecting microscope.

The heart was dissected free from.the great veins, being
careful to leave the auricles of the atria intact. The atria
were difficult to distinguish from the veins. The arteries
were dissected at the point of emergence from.the heart where
the color changes from deep red to white.

The lungs were dissected free of the pleural membranes,
the trachea and the chief bronchi. This dissection was one
of the most difficult to perform uniformly.

The spleen was removed from the surface of the stomach
and cleaned of connective tissue with a sharp scissors.

The liver was removed from the fetus, and the gall bladder
and the cystic duct were removed from the liver before weighing.
The adrenals were dissected from the animal before the
kidneys were removed. The adrenals were cut away from the
connective tissue and associated blood vessels by the use
Of a sharp scalpel. The remaining connective and arterial

tissue was removed by the aid of a dissecting microscope.

The kidneys were removed from.their retroperitoneal

.1”.

position, and the arteries, veins and ureters were cut at the
hili. The renal capsules were left intact.

The testes were severed from the epididymes and vas
deferentia while the ovaries were freed from.all encompass-
ing tissue.

Preparation of tissue for enzyme analysig

After several unsuccessful attempts to produce a uniform
homogenate of fresh tissue, Thompson (1960) suggested the
use of a dental amalgamator. The procedure is outlined be-
low.

Approximately a 1 gram sample or less of thawed wet
tissue was weighed directly into a 2 milliliter tared plastic
capsule. The tissue in the capsule was shredded with a sharp
scissors and weighed again and the tissue adjusted until

the sample weighed. 1 t 0.01 grams. After removal to a

cold room.(2° 0.), a small amount of ice water and a hard
plastic bead were placed in the capsule. Finally, the cap-
sule was capped with a plastic cap and placed on the dental
amalgamator. The tissue was agitated for 3 minutes after
which the homogenated tissue was quantitatively removed from
the capsule, bead and cap by washing with water and decant-
ing into a calibrated centrifuge tube. Ice water was added
to the tube to dilute the original tissue homogenate 10:1.
The tissue was then suspended by gentle hand agitation, after
which the enzyme suspension was centrifuged in.the cold room
for 15 mdnutes in an Ivan Sorvell Type M centrifuge at ap-
proximately 3000 rpm. All the tissue samples were diluted

bl f

at"

o
r-s
('0'

F‘.‘
v‘va

rs

-ua-

to the same proportions in order to facilitate comparison
of the enzyme assays on a wet tissue basis.

When enough tissue remained after sampling for enzyme
analysis, a 2nd sample was taken for dry matter analysis.
The samples were dried 12 hours in a forced draft oven at-
105° C.

Lactase assay

The procedure for the lactase assay was performed ac-
cording to Colowick and Kaplan (1955) using o-nitrophenyl-
fL-D-galactopyranoside as a substrate. The procedure was
modified to produce a pH of 5.6 using an acetate buffer in-
stead of a buffered medium of pH 7.25 as reported in the
literature. The procedure was chosen over the procedures
onHielskov (1951) and Walker (1959) which involved titri-
metric techniques. The o-nitrophenyl-fiLD-galactopyranoside
substrate allowed spectrophotometric analysis of the activity
of the enzyme.

The lactase activity on the wet tissue basis was cor-
rected to a dry matter basis and expressed as mM of o-
nitrophenol released in 15 minutes per 100 milligrams of
dry tissue.

The procedure at all fetal ages and for the newborn pig
was performed as follows:

(1) A standard curve for o-nitrophenol was obtain-
ed by preparing a working standard of 0.1 mM o-nitrophenol.
The optical densities of standard solutions of 0.01 mM to

0.1 mM were measured in a Bausch and Lomb "Spectronic 20"

-h9-

spectrophotometer.

(2) The reaction mixture for the lactase assay
consisted of 3.5 milliliters of a 0.2 M, pH 5.6 acetate buf-
fer (previously determined. to be the optimum pH), 0.5 milli-
liter of 0.01 M o-nitrophenyl-p-D-galactopyranoside and 1.0
milliliter of the enzyme preparation of 1 gram wet tissue
per 10 milliliter of water.

Procedure for alkaline phosphatase
The procedure for the intestinal alkaline and acid phos-

 

phatase assay was a modification from the clinical procedure
commonly used for serum alkaline and acid phosphatasel. The
modified procedure is as follows:

(1) One milliliter of the alkaline buffered sub-
strate (equal proportions of a pH 10.5, 0.1 M glycine buffer
and a p-nitrophenyl phosphate substratel) was pipetted into
a test tube. A blank was similarly' prepared. The tubes
were placed in a u0° C. constant temperature water bath.

(2) Noting the exact time, 0.1 milliliter of the
enzyme containing solution was pipetted into the reaction
tube. The enzyme containing solutions were diluted to in-
sure an enzyme concentration which would produce products at
a rate that could be measured without changing the time of
the reaction. The tissue prepared from.term pigs was diluted,
therefore, 100:1, the 93 day tissue was diluted 10:1 and the
729 51 and 30 day tissues were not diluted at all.

¥

I "Sigma 10h Phosphatase Substrate", Sigma Chemical
Company, St. Louis, Mo. Sigma Tech. Bull. 10h. 1958

- 50.-

(3) After 30 minutes, 10 milliters of 0.02 I! HaOH
were added to each tube to step the activity. Mixing was
accomplished by inversion. .

(h) The optical density was recorded for the sample
with the spectrophotometer set at hlo millimicrons and zero-
ed with the reference blank.

(5) Two drops of concentrated HCl were added to the
reaction tube to remove the yellow color due to p-nitrophenol.
The new optical density was compared to the original measure-
ment. This reading thus corrected for any interfering color
contributed by the tissue homogenate and was subtracted from

the 1st.

Procedure for acid phosphatase
(1) One milliter of the acid buffered substrate

(equal portions of pa n.8, 0.9 M citric acid buffer and the
P'nitrophenyl phosphate substrate solution}) was added to a

tube 0 7
(2) Into the substrate and buffer, 0.2 milliters

Of the enzyme containing extract was pipetted, noting the

exact thme.

(3) Exactly 30 minutes later the enzymatic action

was stopped by adding h milliliters of 0.1 N NaOH. The Stflp

P16 was mixed by inversion.
(h) The Optical densities of the samples were de-

termined using the Bausch and Lomb "Spectronic 20" set at

 

—“ I» bstrate" Sigma Chemical
"Sigma 10h Phosphatase suSigma Tech. Bull. 10h. 1958

Company, St. Louis, Mo.

*h. 7‘

\
Kw-

-51-

M0 millimicrons using 0.1 N NaOH as a reference blank.

(5) To correct the optical density for color con-
tributed by the tissue homogenate, 0.2 milliliter of diluted
tissue extract was added to 5 milliliter of 0.1 N NaOH and its

optical density checked against the NaOH reference blank.

 

RESULTS AND DISCUSS ION

§§§ect of litter size on fetal grpgth

The purpose of correlating litter size with measures
of fetal growth.was to determine if litter size reduced the
dimension of a particular measure at any of the fetal periods
studied. If the correlations were negative, one could pre-
sume.competition for nutrients from maternal origin had taken
place. Two assumptions, which are self-evident, were made.
The 1st assumption was that nutrients, during the fetal per-
iod, reach the fetus by passage across the fetal membranes:
the 2nd assumption was that larger litters compete to a
greater extent than smaller litters for these nutrients.
Pomeroy (l960c) has suggested that competition for nutrients
between fetuses within litters starts after the mid-point of
pregnancy when the placenta has reached its maximum size.

The correlations derived from the research reported here
are presented in Table 5. Even though no consistent pattern
was noted for a particular measure such as height, crown-
rump length, bone length or organ weight, it was observed
that a greater proportion of the correlations of litter size
versus each measure were negative at h of the 5 periods
studied. The majority of the correlations were negative at
hS days, 51 days, 93 days and birth while at 72 days the
mnjority were positive. The percentage negative correlations

at each of the periods studied are presented in Table 6.

-52..

- 53 -

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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-5u-

Table 6. Percentage negative correlations of litter size
with measures of fetal growth

 

Period _ Percent negative correlations
1&5 days 77%

51 days 76%

72 days 15%

93 days 60%

Birth 58%

 

The positive correlations at 72 days, as well as the
negative correlations at 93 days and birth could be explain-
ed by Pomeroy's theory; i.e. competition between fetuses
starts at mid-pregnancy. The larger proportion of negative
correlations at hS and 51 days could not be explained by this
reasoning, because low insignificant correlations would be
eXpected before mid-pregnancy.

Means and variability data of litter size at each fetal
period are Shown in Table 7. Since the number of litters
used at each period were limited, no valid conclusion of the
effect of thme of pregnancy or variability of litter size

could be made. ‘

Summary: The correlation of litter size with measures
of fetal growth was not appreciably influenced by fetal age.
The data did reveal a smaller proportion of negative cor-
relations for the measures at 72 days following breeding

than in any other period studied.

'55"

Table 7. Mean litter size and variability of litter size

 

Days
Birth Birth Birth

30 1 +12 93 Gilts Sows Combined)
number of if 3 7 5 E H 3 H 7
63 51+

 

 

litters

Number of 32 59 51 37 h5 82
fetuses
Mean litfer 1006 900 1008 1&075 1207 1203 1103 10.6
s so
Standard 1058 1.80 Sosa 1.52 107“ 1023 209a 2038
deviation
Standard 0.91 0.69 0.85 0.76 0.87 0.71 1.55 0.85'

error
Relative stand- 806 609 702 901 608 508 13.7 702
ard error (

 

Effect of sex of the fetus on fetal growth

The data illustrating the relationship of sex of the
fetus to fetal growth at each of the periods studied are
found in Appendix Tables 3, h, 5,.6 and 7. Table 8 lists the
t values of data obtained from males compared with that de-
rived from.females. Sex of the fetus had little effect on
fetal growth at any of the fetal periods or at birth, al-
though males tended to be slightly heavier, longer and the
organs larger at all periods. Only the fetuses at us days
exhibited a significant difference between sexes for weight,
crown-rump, head width, humerus length measured from head
to condyle, and humerus‘length ‘measured from.tuberosity to
condyle. Differences were not eXhibited at the other periods
BXOOPt in the case of the spleen and the left and right a-
drenal which.were heavier in.males than in females at 72 days.

The gonads were significantly heavier in males than in.females

- 56 -

 

 

 

 

 

 

 

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.no
HIEOH nuance seams

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Hdb
a I 0
weaken
H1909 m
an 6330
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0% on
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no nos
HHM .

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8

at all periods.
Summary:

-57-

Although the differences were generally not

significant for the effect of sex, the male fetuses and pig-

lets tended to be slightly heavier, longer and the organs

heavier at each of the periods.

Effect of fetal_ageon fetal growth

Means, standard errors of the means and

relative standard errors of the means.

Mean values, standard errors of the means and relative

standard errors of the means at each fetal period and at

birth are included in the Appendix Tables. The data are pre-

sented as follows:

Appendix Table
Appendix Table
Appendix Table

Appendix Table
Appendix Table
Appendix Table
Appendix Table
Appendix Table

1. Development of the pig at birth
(Yorkahire gilts)

2. Development of the pig at birth
(second litter sows)

3. Development of the pi at birth
combined)

a. Development of the fetal pig at 93 days
5. Development of the fetal pig at 72 days
6. Development of the fetal pig at 51 days
7. Development of the fetal pig at #5 days
8. Development of the fetal pig at 30 days

'These tables list the means, standard errors_and relative

standard errors of the means (RSE) for males and females at

each fetal period.

Since the effect of sex, as previously

stated, was not significantly different, the data for males

and females were combined. Means, standard errors and RSE's

were then calculated for the combined data.

 

pres

eval

EN

65:

:1

V"

D

 

- 53 -

The relative standard errOr of the mean (RSE) was ex-
pressed as the percentage which the standard error comprises
of the mean, Snedecor (1953) described a similar method of
evaluating standard deviations. The term.when applied to
standard deviations was called "coefficient of variation" by
Snedecor. Since the relative standard error of the mean
(RSE) in this experiment was expressed as a percentage, the
degree of variation existing in one measure can be readily
compared with another.

These RSE values were therefore used to evaluate measures
of fetal growth by ranking the BBB of several measures from
smaller to larger. The higher ranking measures and their RSE
are listed in Table 9. Also Appendix Table 1 through 8 list
each measure and its respective RSE. These rankings indicat-
ed that skeletal measures were more precise indicators of
age than whole body weight or weight of individual organs.

In general, RSE of measures of a single dissected bone rank-
ed as well as RSE of crown-rump length: however. at MS days
crownerump length was superior to either dissected humerus
or measures of calcified diaphyses as indicators of age.

Summary: Means and standard errors of the means.for
males and females were reported for each fetal period. Since
sex had little effect, data of males and females were come
bined and overall means, standard errors of the means and
relative standard errors of the means were reported for the
combined data. Skeletal measures produced smaller relative

standard errors of the mean than measures of soft tissue or

weight of the whole fetus.
Growth patterns

The patterns of growth as indicated by several of the
measures of fetuses and piglets from Yorkshire let-litter
gilts are presented in the following tables and figures.

Table h lists the anatomical measures performed. The
procedures for the measurement of the organs and tissues were
discussed previously in the section entitled Materials and
Methods.

Table 10 lists the means of the measurements at each
fetal period.

Figure l graphically illustrates the changes in body
weight, head width and crown-rump length as the pig develops.

Figure 2 illustrates the development of the brain, liver
and lungs. Note that the lungs increased in weight only
slightly from.93 days to birth.

Figure 3 shows the change in length of the calcified
diaphyses of certain bones of the fore limbs.

Figure A depicts the same change in the bones of the
rear limbs. Note that all skeletal measures were linear
with age.

Figure 5 illustrates the development of the heart and
kidneys plus the dissected humerus (total length measured
from tuberosity to condyle). Note again that this skeletal
measure was nearly linear with age.

Figure 6 shows the change in weight of the spleen, and

male and female gonads. The spleen and female gonads were

Afi~n

: wk - mi. Dcm 7M. .4. P.

u.- ...a We wt.

 

- 60 -

Table 9a. Comparison of relative standard errors of the
mean at birth - from Yorkshire gilts

 

 

Measure RSE Rank

Head width 0.1; 1

Brain weight 1.9 2

Humerus (H - 0) length 2.3 3
Crown-runm len th 2.9 h

Humerus. (X-ray length 3.5 5

Humerus (T - 0) length 3.6 6,7,8 (i210)
Ulna (X-ray) length 3.6 6,7,8 (tie)
Femur (X-ray) length 3.6 6.7:»8 (1516)
Radius (X-ray) length 3.7 9

Fibula (X-ray) length h..0 10

 

Table 9b. Comparison of relative standard errors of the
mean at birth - from second litter Duroc sows

Measure

 

RSE Rank

Humerus (H - 0) length 1.2 1,2 (tie)
Radius (X-ra ) length 1.2 1,2 (tie)
Femur (X-ray length 1.3 3

Head width 1.1:, LL.S (tie)
Fibula (X-ray) length 1.u %.5 (tie
Humerus (T " 0) length 105 :798 (tie)
Humerus (X-ray) length 1.5 6,7,8 (tie)
Tibia (X-ra ) length 1.5 6.7.8 (tie)
Ulna (X-ray) length 1.9 9,10 (tie)
Metatarsal (3 or )4) length 1.9 9.10 (tie)

 

-61-

Table 9c. Comparison of relative standard errors of the
means at birth - combined data of Yorkshire gilts
and secondélitter Duroc sows ‘ -

 

Measure RSE Rank
Humerus (H.- 0) length. 1.3 1,2 (tie)
Ulna (X-ray) length 1.3 1.2 (tie)
Humerus (T - 0) length l.u 3,h (tie)
Radius (X-ray) length 1.h 3,? (tie)‘
Humerus (x—ray) length 1.5 5. (tie)
Femur (X-ray) length 1.5 5,6 (tie)
Head width 1.6 7,8 (tie)
Brain weight 1.6 7,8 (tie)
Fibula (X-ray) le th 1.7 9
Metatarsal (3 or u (X-ray) 1.8 10

length

A

Table 9d. Comparison of relative standard errors of the
'means at 93 days - from Yorkshire gilts

 

 

 

Measure ESE Rank “
Humerus (n - 0) length 0'3 1

Head width 8‘; §

Crown-rump len th °

Humerus (T - 0? length 1'2 ”'5 (tie;

Ulna (X-ray) length 1.2 2:5 (”1°
Ischium (X-ray) length 1’3 8 (ti )

Ilium (X-ray) length 1‘” 7’8 (tie)

Femur (X-ray) length 1’“ 7' e

Tibia (X-ray) length {'3 20

_Pituitary weight

 

- 62 -

Table 9e. Comparison of relative standard errors of the
means at 72 days - from Yorkshire gilts

WM

 

Measure RSE Rank

Humerus (T - C) length 0.1 l

Humerus (H - C) length 0.8 2.3 (tie)

Radius (X-ray) length 0.8 2.3 (tie)

Head width 0.9 h

Crown-rump len th 1.2 5,6 (tie)
Humerus (x-ray length 1.2 5,6 (tie)

Brain weight 1.3 6.7.8.9.10 (tie)
Ulna (x-ray) length 1.3 6,7,8,9,10 (tie)
Femur (X-ray) length 103 6979839910 (tie)
Tibia (X-ray) length 1.3 6,7,8,9,10 (tie)

Table 9f. Comparison of relative standard errors of the
means at 51 days - from Yorkdhire gilts

 

 

Measure RSE Rank

Head width 0.5 1
Humerus (H - C) length 0.7 2
Crown-rump len th 1.0 3
Humerus (T - 0% length 1.3 8.5 (tie)
Humerus (X-ray) length 1.3 %,5 (tie)
Femur (X-ra ) length l.h

Ulnfi (X-ray length 1.5 7

Tibia (X-ray) length 1.6 8

Radius (X-ray) length 1.8 9

Heart weight 2.u 10

 

-63-

Table 9g. Comparison of relative standard errors of the
means at h5 days - from crossbred first litter

 

 

 

 

 

gilts
W M
Measure RSE Rank
Crown-rump length 2.u 1
Head width 2.6 2
Humerus (T - 0) length 3.1 3
Humerus (H - 0) length 3.2 u
Ulna (x-ray) length 5.9 5
Humerus (X-ra ) length 7.3 6.? (tie)
Radius (X-ra length 7.3 6,? (tie)
Femur (X—ray) length 8.3 8
Body weight 8.7 9
Tibia (X-ray) length 10.2 10

Table 9h. Comparison of relative standard errors of the
means at 30 days - from.Yorkshire gilts

 

 

 

 

\\

r —- _ ~ —-=——
“l -,

 

 

 

Measure RSE Rank __
Crown-rump length 1.2 1
Head width 2.9 2
Body weight 3.3 3

 

- 6h -

 

 

 

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93 Birth

AGE, DAYS

1 pigs -
H e 1. Growth atterns of Yorkshire feta
8111‘ Meight,pcrown-rump length, head width.

 

 

 

:35 -
E? 25
5
H15“
E:
5
51 72 93 Birth

AGE, DAYS

Figure 2. Growth.patterns of Yorkshire fetal pigs -
Brain, liver, lung.

LENGTH, MM.

30

[0
O

H
C

Figure 3.

LENGTH, MM.

30

N
O

H
O

PupuMIh.

 

 

- 66 -

  
 

 

Humerus
/, Radius
'// Metac.
h- ,/
x” '/'
o/
/
51 72 93 Birth
AGE, DAYS

Growth patterns of Yorkshire fetal pigs -
Humerus, ulna, radius, metacarpal (3 or a
ma. 2 01‘ S).

U

 

 

 

L
VL 1 A 1
51 72 93 Birth
AGE, DAYS

Growth patterns of Yerkshire fetal pigs -

Feaur, tibia fibula, ilium, ischium, calcaneous
metatarsal (5

or u and 2 or 5).

-67-

   

 

 

Heart
:5
C)
Es > i,’ Hunerus
z I
3
Xidne
83 - ./’ y
3
.1 ’
. i’,.
P Lv~+— - . .
51 72 93 Birth

AGE, nus

Figure 5. Growth patterns of Yorkshire fetal pi s -
Heart, humerus (tuberosity to condyle , humerus
(head to condyle) and left and right kidneys.

 

 

Spleen
700*
g 500
E .
.-. 300 ,’ Gonad, m.
g
100} ””1253? .... _. Gonad, f.
w‘ "— . I“

AGE, nus

Figure 6. Growth patterns of Yorkshire fetal pigs -
Spleen, left and right gonad of the male,
left and right gonad of the female.

  
    

 

 

Thyroid
150'
. 120 L Adrenal
I
3 ,
. /
E5 90 L /
/
H I
I
9'.“ 60 Y ,
I
30 . ’ /‘ ,- Pituitary
E’" .1 L A
“[51 72 93 Birth
AGE, DAY!

Figure 7. Growth patterns of Yorkshire fetal pigs -
‘I'hyroid, left and right adrenal and pituitary.

-68-

lighter at birth than at 93 days.

Figure 7 depicts the development of the thyroid, adre-
nals and pituitary. The growth of the thyroid and pituitary
were nearly linear from 51 days to birth. The adrenals in-
creased appreciably in weight the last 3 weeks of the gesta-
tion period.

.Summary: Growth patternsof fetal development of York-
shire fetuses were determined for measures of growth of the
skeleton and organs. The growth pattern for measures of the
skeleton were generally linear with age from 51 days to birth.
The adrenal made a considerable increase in weight the last
3 weeks of fetal life.

Correlation of measures_with fetal age

Correlations of certain representative measures with
age for the data from.Yorkshire gilts are presented in Table
11. All coefficients presented were highly statistically
significant; however, measurements which represented length
of certain portions of the skeleton were more highly cor-
related with age in utero than were measures which represent-
ed soft tissue growth. For example, correlation coefficients
'of well over 0.95 were established for the skeletal measures,
except head width, while correlation coefficients of'0.6h to
0.90 were found for soft tissue weights.

Even though one would expect that the weights of the
organs illustrated would be highly related to age, the author
did not expect high correlation for both calcified and soft

tissues. 'Previous experience suggested that any circumstance

-69-

Table 11. Correlations of age with measurements of fetal

 

 

 

growth!
Measure and period of study r
Body weight (30 days to birth) .830
Crown-rump length (30 days to birth) .98h
Head width (30 days to birth) .80h
Length of ---
Humerus, H-C (51 days to birth) .983
Humerus, T-C (51 days to birth) .983
Femur, X-ray (51 days to birth) .980
Humerus, X-ray (51 days to birth) .977
Weight of ---
'Liver (51 days to birth) .75u
Spleen (51 days to birth) .830
L. Kidney (51 days to birth) .898
DR. Kidney (51 days to birth) .903
L. Adrenal (51 days to birth) .67?
R. Adrenal (51 days to birth) .799
L. Gonad, female (51 days to birth) .6h3
L. Gonad, male (51 days to birth) .773
R. Gonad, female (51 days to birth) .66?
R. Gonad, male (51 days to birth) .795
Thyroid (51 days to birth) .906
Heart (51 days to birth) .828

 

‘ Datafiof fetuses from.Yorkshire gilts at 30 days,

51 days, 72 days and 93 days post-breeding and at

-70-

resulting in temporary or prolonged inanition would cause a
quantitative change from normal in soft tissue much more
quickly than in.the skeleton. It is suggested then, that
either measurement of a single dissected long bone or of an
X—ray of a long bone would serve as a better estimate of age
than.measures of soft tissue.

Historically, estimations of fetal age of animals of
unknown conception date had been based on crown-rump length.
It 'is suggested that there are several reasons why this
measurement may be somewhat undependable even though cor-
relations with age in.this study were very high. First of
‘ all, repeat measurements were nearly impossible to exactly
duplicate because of changes in body position and errors
were thus more likely than when measuring a single bone or
weighing an animal or a single organ. The crown-rump
measurement involved soft as well as skeletal tissue which
may vary considerably in quantity between fetuses of the same
age. However, measurement of crown—rump was a simple measure
to perform, and secondly, its measurement early in fetal life
could be accomplished easier than dissection of an organ or
bone. Also, the calcification of the diaphyses of the long
bones at 30 days was not sufficient to permit their use that
early in fetal life.

Summary: Correlations of fetal age with measures of
fetal growth were highly significant. Values for skeletal
measures, except for head width, ranged above 0.95, while

correlations with measures of soft tissue including weight

- 71 -

of the whole fetuses ranged from 0.6h to 0.90.

Predicting fetal age from fetalggrowth data

Predicting equations suitable for estimating ages of
fetuses from length of bones and the crownsrump length are
presented. Accompanying each equation is the correlation
of the measure with age and the standard error of estimate
for the equation.

The equations were derived from data of fetuses from
first-litter Yorkshire gilts. The measures selected for
development of these equations met several important cri-
teria. First of all, the measures were easy to obtaini
secondly, they were consistently duplicated; thirdly, the
measures were of skeletal growth which possessed a higher
correlation with age than measures of the soft tissues;
and fourthly, the measures possessed low relative standard
errors of the means at each of the fetal ages.

The following equations describe bone growth of the
fetal pig from 51 days post-breeding to birth:

Humerus (tuberosity to condyle, dissected).
y = 29.00 + 1.576x

x = length of dissected humerus, mm.
y = age, days

ry,x = 0.98

Syox g 0.90
Humerus (head to condyle, dissected).
y = 31e31 + 1.6140}:

x = length of dissected humerus, mm.
y = age, days

-72-

1' . = Oe98
8%.: a OeBO

‘Humerus (calcified diaphysis, x-ray).

y s.uo.15 + 1.87:

x = length of calcified diaphysis, mm.
y = age, days

ry,x = 0.98
8y.x g 0e88

Femur (calcified diaphysis, X-ray).

y = hl.6o + 1.87x

x 8 length of calcified diaphysis, mm.
y = age, days

0.98

r ex
y 0.7?

3y.x

The following equation represents the changes of the

crown-rump measurement from 30 days following conception to

birth:

Summary:

“ 21.07 + 0.311x

:4
I

x = length from crown-rump, m.
y = age, days

0.98
0.73

Five estimating equations were suggested for

predicting ages of fetuses from Yorkshire, first-litter gilts.

The measures used for developing estimating equations were

selected because they were: (1) anally measured, (2) readily

duplicated, (3) highly correlated with fetal age and (u)

possessed a low relative standard error of the mean at each

fetal period.

Standard.errors of estimate of the predicting

equations were all below $1.0 day.

-73-

Estimation of time of appearance, location.and_guantity

of intestinal lactase and alkaline and acid os atase

The distribution of intestinal lactase and alkaline and
acid phosphatase at birth as determined in.this study is swa-
marized in Table 12.

Assays revealed that the cranial half of the duodemmm
and the caudal 3rd of the jejunumsileum.were significantly,
lower (P<0.05) in lactase activity than the other portions
of the gastro-intestinal tract when the data were examined
using the Studentized range test. Therefore, the middle
portions of the small intestines exhibited more lactase
activity than the tissue near the stomach or colon. The
stomach and colon did not exhibit lactase activity at any of
the fetal periods.

A shmilar pattern was noted for alkaline phosphatase
activity at.birth. The results showed that the activity was
significantly less in the entire duodenum and in the caudal
3rd of the Jejunumeileum than in the cranial and middle 3rds
of the Jejunumpileum. Again, there was no activity in the
stomach or colon.

Acid phosphatase assays of tissue from these 5 areas
of the mmall intestine revealed a similar tendency, but only
the cranial portion of the duodenum was significantly lower
in acid phosphatase activity than the other areas. There
was no acid phosphatase activity in the stomach and colon.

Although there were significant differences in dis-

tribution.in the small intestine of the 3 enzymes when studied

- 7h -

Table 12. Enzyme distribution - birth

 

 

Lactase‘ Alk. PhosbP Acid Phos.b
Duodenum, Cran. .19 %l 5.8
Duodenum, Caud. .38 7 8.0
JOJe-Ile\m, Crane .36 1'45 908
JeJ.-Ileum, Mid. .36 17 9.h
JeJ.-Ileum, Caud. .25 5 9.h

 

a mM o-nitrophenol released in 15 minutes per 100
milligrams of dry tissue. -

b1mH p-nitrophenol released in 30 minutes per 100
milligrams of dry tissue.

Table 13. Fetal development of lactase, alkaline and
acid phosphatase activity

 

Age (days) SD.H. Lactase‘ Alk. l’hos—b Acid Phos.by:

n x n X x
30 3' .20 35 E.E 38 5.8
51 13.“. 6 .20 7 2e 7 6e6
72 13.8 35 .10 33 2.h 36 5.1
93 nus 27 .21, 23 8.0 25 5.8
Birth 17.2 51 .31 53 10h.6 53 8.8

.._¥

a mu of o-nitrophenol released in 15 minutes per 100
milligrams of dry tissue.

b mM of p-nitrophenol released in 30 minutes per 100
milligrams of dry tissue.

c Pooled data of 8 fetuses of 3 litters.

-75-

at birth, the area differences in enzymatic activity were not
significant at 93 days except for lactase whieh was signifi-
cantly lower in the duodenum than the jejunum.and ileum.(Ap-
pendixVTable 10). At 72 days the duodenum and caudal third
of the Jejunumrileum.were lower in alkaline and acid phos-
phatase activity than the cranial and middle portions of the
Jejunum-ileum (Appendix Table 11). The activity for lactase
at 72 days also indicated a simdlar pattern, however the
differences were not significant. 1

Table 13 illustrates the change in mean intestinal en-
zyme activity with age.. These means include all 5»areas of
the intestine. In each case, for the 3 enzymes studied, the
activity at birth was significantly greater than the values
at 93 days. Also, alkaline phosphatase activity was signi-
ficantly greater at 93 days than at 72 days. The increase
in activity from 93 days to birth for lactase was 1.3 fold,
for alkaline phosphatase was 13 fold and for acid phosphatase
was 1.5 fold.

The appearance of alkaline phosphatase followed a pat-
tern similar to that reported by Moog for the chick embryo
and mouse fetus-(Moog, 1950: Moog, 1951; Moog and Wenger,
1952; Moog, 1953). Acid phosphatase activity in this study
did not increase as rapidly as that of alkaline phosphatase
which was.similar to findings reported by Moog (1958).

A complete record of the intestinal enzyme assays per-
formed in this study may be found in Appendix Tables 9, 10,
11, 12 and 13.

-76-

Summary: At birth the caudal half of the duodenum.and
the cranial two-thirds of the jejunum-ileum.possessed great-
er lactase and alkaline phosphatase activity than did the
remainder of the gastro-intestinal tract. Acid phosphatase
was more uniformly distributed through out the small intest-
ine than.either lactase or alkaline phosphatase, but activity
was significantly less in the cranial duodenum.

The enzymatic activity was significantly greater at
birth than at any of the fetal periods for lactase, alkaline
phosphatase and acid phosphatase. Alkaline phosphatase act-
ivity was also significantly higher at 93 days than at 72
days.

Time patterns for the rise in activity of alkaline and
acid phosphatase, which had been reported for other fetal

species, were confirmed for the fetal pig.

SUMMARY

The objectives of this study were to provide information
in 2 areas concerned with the development of the swine fetus;

1. Measures of body size, organ weight and skeletal
development which would promote better understanding of the
relationships between the anatomy and physiology of growing
fetal structures and whiCh would allow for more accurate
estimation of age of fetuses of unknown conception date.

2. Estimation of the time of appearance, location
and concentration of intestinal lactase and acid and alka-
line phosphatase.

Fetal and newborn pigs were obtained from 19 first-litter
Yorkshire gilts which were selected for uniformity of size
and age. Supplementary information.was gathered from fetuses
from»? Hampshire x Duroc gilts and piglets from h.Duroc sec-
ond-litter sows. At appropriate estrous periods, the gilts
were bred to 2 Yorkshire boars, 1 serving a gilt once on the
1st day followed by the 2nd boar on the succeeding day. If
further estrus were not observed, the gilts were considered
Pregnant and caesarian sections were performed 30, 51,72 or
93 days post-breeding or the gilts were allowed to farrow
naturally. The crossbred gilts were slaughtered at h5 days
and the fetuses removed from.the excised uteri.

Body weights, crown-rump lengths and head widths across

the parietal bones were measured immediately after removal

- 77 -

-73-

from.the uterus or just following birth. The weights of in-
ternal organs and the lengths of the humeri were obtained
immediately subsequent to dissection. The other length
measurements were obtained from X-ray photographs and were
measures of the calcified diaphyses of the bones of the ap-
pendages.

Sections of the gastrointestinal tract were separated,
identified and frozen in dry ice where they were stored un-;
til enzyme assays could be performed.

Correlations of litter size with measures of fetal growth
were accomplished at each fetal period. Size of litter
did not consistently affect the dimension of any particular
measure. The data, however, did reveal a smaller proportion
of negative correlations for measure versus litter size at
72 days following breeding than in any other fetal period
studied or at birth.

Although the differences for most measures between.males
and females were generally not significant, male fetuses and
male piglets were slightly heavier, longer and the organs
heavier at each of the periods.

Means, standard errors of the means and relative stand—
ard errors of the means for both sexes and for the sexes com-
bined were presented. Skeletal measures gave smaller relative
standard errors than measures of soft tissue or weight of
the whole fetus.

Growth patterns plotted from.various measurement means

Of fetal and newborn pigs were presented. Measures of

.‘79-

skeleton were generally linear from 51 days to birth. The’
adrenals made the majority of their grewth the last 3 weeks
of fetal life.

A Correlations of measures of fetal growth with age post-
breeding were highly significant. Coefficients of age cor-
related with measures of skeleton ranged above 0.95 except
fhr head width, while measures of soft tissue ranged from
0.6h to 0.90.

Five esthmating equations were suggested for use in
predicting ages of fetuses from.Yorkshire first-litter gilts.
The particular measures selected met several important cri-
teria, which were that they; (1) be easily measured, (2)
be consistently duplicated, (3) be highly correlated with
fetal age and (h) possess a low relative standard error of
the mean at each fetal age. Standard errors of estimate of
the predicting equations were all below :1.0 day.

At birth, the middle portion of the small intestine
possessed a greater enzymatic activity for lactase and al-
kaline and acid phosphatase. Acid phosphatase was more
uniformly distributed throughout the small intestine than ‘
either lactase or alkaline phosphatase except in the cranial
duodenum.where its activity was lower.

Activity of these 3 enzymes were significantly greater
at birth than at any of the fetal periods studied. Alkaline
phosphatase activity was also significantly higher at 93
days than at 72 days.

Time patterns for the appearance of alkaline and acid '

-80-

phosphatase which had been reported for other fetal species
were confirmed for the fetal pig.

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- 89 -

 

 

 

 

».m oe. mm.a:oo mo.~m m.e~m ca .me..eemaes assesseaa
o.H me.o sum .asmma mo.mn 30.0mm as .am .eewne: aseem
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o.o m .o moee.mee oe.e 10.90 as .sw .eewnee eeeem
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- 102 -

Appendix Table 9. Enzyme analysis - birth

A. Alkaline phosphatase
mM of p-nitrophenel per 30 min. per 0.01 mg. of wet tissue.

11 2x 2x2 3E 33
1. Duodenum, cranial . . . .
2. Duodenum, caudal 8 1.21 0.22gg 0.15 0.03
3. J. I., cranial 12 3.05 0.8617 0.25 0.03
%. J. I., midgli if {.63 g.§5hg 0.30 0.03
0 Jo Is. 0811 a e e 0.10 0.02
Total 33 5.73 . 0713 ‘5762

p a 13.h2**; SE! = 0.02; number 3 and number n
are significantly different from all others, but not
from each other.

B. Acid phosphatase

an of p-nitrophenol per 30 min. per 0.16 mg. of wet tissue.

a 2x 2x2 it SE
1. Duodenum, cranial

O O .1 O
2. Duodenum, caudal 8 1.76 0.3708 0.22 0.0h
3. J. 1., cranial 12 3.21 0. 781 0.27 0.01
%. J. I., middle 12 3.;2 0.3556 0.26 0.02
0 Jo I. caudal ll 2. 0 0 0.26 0.0
'i‘otal 33 332733 0723 070%

F = u.uh**; SEE = 0.02; number 1 is significantly
different from numbers 3, u and 5 but not from number.2.

*—

C. Lactase
mm of o-nitrophenol per 15 min. per 100 mg. of wet tissue.

11 2x 21:2 if SE
1. Duodenum, cranial. . 1 0 0.

2. Duodenum, caudal 7 0:h62 0.03h83 0.066 0:010

3. J. 1., cranial 12 0.738 0.00775 0.062 0.003
1;. J. 1., middle 11 8.6 7 0.013677 0.062 0.003

S. J. 1., caudaL. 11 o 0221 0.0 0.002
Total 30 2%? 6.33%; 0. 0".0611

F a 11.88“; See . 0.004; numbers 1 and s are signi-
ficantly different.rrom.numbers2, 3 and u but not each other.

- 103 -

Appendix Table 10. Enzyme analysis - 93 days

 

 

A. Alkaline phosphatase
mH of p-nitrophenol per 30.min. per 0.1 mg. of wet tissue.

N 21: xx? x SE

1&2. DuOdCM‘ g 60E 606326 aeiI 6062
3. Jo Io, crani‘l 1000 001356 0013 0002
15,. J. 1., middle 1. 0- 0.0509 0.10 0.03
. J. 1., caudal 2; 0. 2 0.1212 0.12 0.02

P 8 0.21; SEE = 0.010

 

B. Acid phosphatase
an of p-nitrophenol per 30 min. per 0.16 mg. of wet tissue.

N 22x 8X2 1 SE

1&2. 13110de e e e e
3. J. 1., cranial 9 1.35 0.2229 0.15 0.02
'40 J0 Io, middle 5 0066 0.0890 0013 000“.
5. J. 1., caudal. 6 0.8 0.12%% 0.1 0.01
TOtal I; e e e m

P 3 0.14.7; SE = 00010

C. Lactase
mm of o-nitrophenol per 15 min. per 100 mg. of wet tissue.

_ 0 2x 20:?- X SE
1&2. Duodenum! . . . .
3. J. 1., 61-60161 10 0.395 0.016753 0.01.0 0.00

S. J. 1., caudal 6 0.20 0.00 0 0.0%E 0.00%
Total. ‘27 ‘5:§8% Utajgétfi 0. .
F =~23.o7**; SEE = 0.003; number 1 and 2 are sign-
ificantly different from.nnmbers 3. h and 5.

a The data includes samples from.the cranial and
caudal portions.of the duodenmm.

-1014-

Appendix Table 11. Enzyme analysis - 72 days

A

A. Alkaline phosphatase
mM of p-nitrOphenol per 30 min. per mg. of wet tissue.

a . 2x 12 s
1&2. DUOdOHUM‘ e e e e
30 J. Io, cranial 7 2.62 102180 e

0 3 0.07

go go is, misgli lg 20 86 ieggs6 80%; 0082
e e e 08 a e O.

6.01 11"3 3.0263 05%. 076;

F = 22.30**; SEE 8 0.05; 1 and 2 are significantly
different from 3 and h, but not 5.

w—W

B. Acid phosphatase
an of p-nitrophenol per 30 min. per 0.16 mg. of wet tissue.

11 2x 2x2 1 SE

1&2. DuOdennmg' 13 ICE; OOI§I1 601T 0001

3. J. 1., cranial. 7 1.02 0.1501 0.15 0.01

he J. 10, middle 6 008“ 0.1212 0.1“ 0001

5. J. 1., caudal 10 0. 0 0.0 6 0.0 0.01
166.1 ‘3‘6 .21 0. 03 0. 2 ‘0'“.002

F = 3h. 56**; SEE =>0. 002; l and 2 are significantly
different.from 3 and u but not 5.

C. Lactase
ms of o-nitrophenol per 15 min. per 100 mg. of wet tissue.

u xx ' z x2 1 ss
1&2. DuOdannmP e e e e
30 J. 10, cranial 7 0015“ 00003388 0022 0002
he J. I,, middle 5 0.100 0. .00223%' .020 .003
l

S. 3.1. caudal 101 0. 0020 .008 .00
'l‘otal 35 0. 5 0&1; 61513253103502
F = 3.975: SEE = 000020

“

i The data includes samples from the cranial and
caudal portions of the duodenum.

- 105 -

Appendix Table 12. Enzyme analysis - 51 days and 30 days

A. Alkaline phosphatase
mu of p-nitrophenol per 30 min. per mg. of wet tissue.

N 21 ’ 2x2 1 SE
51 dEYBa o e e
30 daysb 3 1.001.3573 0.23 0.16

 

B. Acid phosphatase

w of p-nitrophenol per 30 min. per 0. 16 mg. of wet tissue.
3

N x 2x2 1 as
f 51 days“ 0 e e e
30 daysb 3 0.3% 0.0190 0.13 0.01
C. Lactase

mm of o-nitrophsnol per 15 min. per 100 mg. of wet tissue.

2
51 day“ N 2x 2x

0 O O O O
30 daysb 3 .006 .000596 0.016 .002

a The data includes all samples of the small intestine
regardless of location.

b The data are from.pooled samples of 8 fetuses.

Appendix Table 13. Percent dry matter of fetal intestinal

 

 

 

 

 

 

 

 

 

 

 

 

tissue
Period Percent—0 40-—
30 days 13.h (estimated)
51 days 3.% (duplicate analysis)
3% days 13.
days 1%k 5 $0.5
Birth 1 :07 07

a These percentage were used to convert assays to dry
matter basis.

 

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