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EMBRYO PRODUCTION, FREEZING AND TRANSFER
IN THE GOLDEN HAMSTER (MESOCRICETUS AURATUS)

By

Mundhir Taiyeb Ridha

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of AnimaI Science

1983

ABSTRACT

Embryo Production, Freezing and Transfer
in the Golden Hamster (Mesocricetus Auratus)

 

by
Mundhir Taiyeb Ridha

The various developmental stages of hamster embryos (l-cell to
morula) were studied as a model for embryo freezing and transfer. The
survival rate of hamster embryos was examined by three different
methods: 1) normal morphology, 2) trypan blue exclusion and
3) transfer techniques. The three tests were positively correlated.
Viability of hamster embryos was significantly higher in tissue
culture-l99 (TC-199) than Dulbecco's phosphate buffere saline (PBS)
medium. Optimum seeding temperature was above -9°C. Best results
were obtained when hamster embryos were slowly cooled (0.33°C/min) and
thawed (l.5°C/min) compared to rapid thawing (90°C/min). The develop-
mental potential (to 6-day and l4—day old fetuses) of l-cell and 2-cell
embryos was significantly lower than other embryos. Superovulation
resulted in lower implantation rates and a 9 h delay in ovulation in
unfrozen embryos. With unfrozen embryos a higher implantation rate
was observed in TC-199 than PBS and unilateral than bilateral transfer.
Intrauterine transfer of 4- and 8-cell embryos was superior to intra-
bursal transfer. The implantation rates of unfrozen hamster embryos
(Z-cell to 8-cell) were similar in both horns. Migration of l—cell
embryos was significantly higher than 2-cell embryos. The transfer of

7 to 9 embryos per horn or oviduct resulted in a higher implantation

rate than 3 to 6 or l0 to l2 embryos per transfer. No significant
difference was observed between the developmental stages of the embryo
and implantation or pregnancy rates. Similar implantation rates were
noted in young (l4 wk) and mature (26 wk) recipient hamsters. Hamster

embryos were found to be a good model for freezing and transfer

experiments.

To Saeeda and Dukelow

with love and appreciation

ACKNOWLEDGMENTS

I wish to acknowledge those people who encouraged me in performing
this work. Sincerest thanks are expressed to my great-hearted major
advisor, Dr. w. Richard Dukelow, who gave me unlimited support, en-
couragement, and guidance during the entire course of this study.

Thanks and appreciation are expressed to my advisory committee:
Dr. L.G. Clemens, Dr. Pericles Markakis, Dr. R.J. Aulerich and Mr.
Richard Lehnert, for reading the thesis and providing valuable sugges-
tions.

The author's appreciation is expressed to the Department of
Animal Science and Faculty. Thanks are also extended to the graduate
students of the Endocrine Research Unit and our secretary Ms. L.M.
"Bonnie" Cleeves for their encouragement and assistance, and to Ms.
Cathy Allen for her efforts in the preparation of this thesis.

Special thanks are extended to the greatest wife, Saeeda, and
children, Ahmed and Ali, who showed faith, support and patience during

my study.

ii

ACKNOWLEDGEMENTS ...............................................
LIST OF TABLES -------------------------------------------------
LIST OF FIGURES ................................................
INTRODUCTION ---------------------------------------------------
REVIEN OF LITERATURE ...........................................
A. Embryo Freezing ......................................
l. Freezing Principles -----------------------------
2. Freezing of Preimplantation Embryos -------------
a. Media --------------------------------------
b. Seeding Temperature ------------------------
c. Freezing and Thawing Rates -----------------
3. Fertility of Frozen Embryos ---------------------
B. Embryo Transfer --------------------------------------
l. Natural and Superovulation for Embryo
Production --------------------------------------
2. Site of Deposition and Type of Transfer ---------
3. Transfer Media ----------------------------------
4. Age of Recipient --------------------------------
5. Number of Transferred Embryos ...................
6. Embryo Migration ................................
MATERIALS AND METHODS ------------------------------------------
A. Animals ..............................................
B. Embryo Recovery and Handling -------------------------
C. Freezing Techniques ----------------------------------
D Embryo Transfer --------------------------------------
I. Basic Technique of Transfer ---------------------
2 Superovulation and Recipient Synchronization--—-
3. Age of Animals ----------------------------------
4. Number of Embryos Transferred -------------------
5 Embryo Migration --------------------------------
RESULTS --------------------------------------------------------
DISCUSSION -----------------------------------------------------
SUMMARY AND CONCLUSIONS ........................................

TABLE OF CONTENTS

iii

Page

ii

V

vii

TABLE OF CONTENTS (continued) Page

BIBLIOGRAPHY --------------------------------------------------- 80
APPENDIX A
Table 28. Effect of freezing medium on viability of
cow eggs -------------------------------------- 92
Table 29. Viability of frozen-thawed cow eggs following
seeding at different temperatures ------------- 93
APPENDIX B
Publications by the author -------------------------------- 95
APPENDIX C
Curriculum vitae ------------------------------------------ 97

iv

Table

10.

ll.

12.

13.

14.

LIST OF TABLES

Viability of Cooled Embryos after Storage between
lO°C and 0°C ........................................

Viability of Frozen Mammalian Embryos at High
Subzero Temperature .................................

Viability of Mammalian Preimplantation Embryos in
Various Media at Temperature between 25°C and
37°C ------------------------------------------------

Normal Development of Unfrozen Mammalian Embryos
Following the Transfer of Different Embryo
Numbers ---------------------------------------------

Viability of Frozen-Thawed Hamster Preimplantation
Embryos in TC-199 ...................................

Viability of Frozen-Thawed Hamster Preimplantation
Embryos in Dulbecco's Phosphate Buffer Saline
PBS -----------------------------------------------

Fertility of Frozen-Thawed Hamster Embryos in
TC-l99 ----------------------------------------------

Fertility of Frozen-Thawed Hamster Embryos in PBS-—-

Fertility of Frozen-Thawed Hamster Embryos in Two
Freezing Media (PBS vs. Tc-199) .....................

Viability of Frozen Hamster Embryos Following Slow
Thawing l.5°C/min ...................................

Viability of Frozen Hamster Embryos Following Rapid
Thawing 90°C/min ....................................

Fertility of Slowly Thawed Hamster Embryos Following
Embryo Transfer .....................................

Fertility of Rapidly Thawed Hamster Embryos
Following Embryo Transfer ---------------------------

Fertility of Frozen Hamster Preimplantation Embryos
Following Slow and Rapid Thawing Procedures ---------

Page

15

I6

22

26

35

36

37
38

39

49

50

51

52

53

LIST OF TABLES (continued)

Page

l5. The Developmental Potential of Slowly Cooled and Thawed

Hamster Embryos in Either PBS or TC-l99 Containing

l.5 M-DMSO ----------------------------------------------- 54
l6. Implantation Following Unfrozen Embryo Transfer in

Hamster -------------------------------------------------- 56
17. Pregnancy Percentage in Hamster Following Embryo

Transfer ------------------------------------------------- 57
18. Recovery Times of Hamster Embryos ------------------------ 58
l9. Survival of Superovulated Hamster Embryos Following

Embryo Transfer ------------------------------------------ 59
20. Implantation in Golden Hamster Following Intrabursal and

Intraoviductal Transfer of Preimplantation Embryos ------- 6O
21. Implantation in Golden Hamster Following Intrabursal and

Intrauterine Transfer ------------------------------------ 6l
22. Effect of Side of Embryo Transfer on Implantation in

Golden Hamster ------------------------------------------- 62
23. Effect of Uterine Horn on Implantation of Hamster

Embryos -------------------------------------------------- 63
24. Viability of Hamster Embryos in Two Transfer Media

(PBS vs. TC-l99) ----------------------------------------- 65
25. Implantation of Hamster Embryos in Young and Mature

Recipients Following Embryo Transfer --------------------- 66
26. Receptivity of Uterine Environment to the Number of

Transferred Embryos Following Embryo Transfer in Golden

Hamster -------------------------------------------------- 67
27. Embryo Migration in Hamster Following Embryo

Transfer ------------------------------------------------- 68
28. Effect of Freezing Medium on Viability of Cow Eggs ------- 92
29. Viability of Frozen-Thawed Cow Eggs Following Seeding at

Different Temperatures ----------------------------------- 93

vi

Figure

LIST OF FIGURES

Page
Recovery times of hamster preimplantation embryos---- 29
Survival of l-cell hamster embryos seeded at
temperatures between -5°C and -23°C ------------------ 40
Survival of 2-cell hamster embryos seeded at
temperatures between -7°C and -23°C ------------------ 41
Survival of 4-cell hamster embryos seeded at
temperatures between -6°C and -20°C ------------------ 42
Survival of 8-cell hamster embryos seeded at
temperatures between -7°C and -l9°C ------------------ 43
Survival of hamster morulae seeded at temperatures
between -5°C and -15°C ------------------------------- 44

Positive correlation between TpB' and morphologically
normal embryos ....................................... 45

Positive correlation between morphologically normal
embryos and implantation ----------------------------- 47

Positive correlation between TpB' embryos and
implantation ----------------------------------------- 48

vii

INTRODUCTION

Embryo freezing and transfer techniques are useful tools for
producing superior genetic stock in domestic animals. These techniques
have been used to examine maternal-embryonic interaction and other
aspects of implantation. The embryo itself is a special kind of organ;
unlike most other cell types, it consists of a group of undifferentiated
cells varying in size during early cleavage and having the potential to
give rise to a complete new individual. Since the successful storage
of mouse embryos in liquid nitrogen (Whittingham, gt_al,, 1972; Nilmut,
l972) the techniques have been improved and applied to the storage of
other embryos. Although the basic technique for embryo freezing is
similar for all species, factors such as the rate of cooling and
thawing, cell permeability, level and kind of cryoprotectant, seeding
temperature, final storage temperature, cell size, stage of embryonic
development and species sensitivity must be considered.

Unfrozen and frozen-thawed preimplantation embryos can be trans-
ferred to recipient females either surgically or nonsurgically. Ad-
vanced stages of embryos (morula and blastocyst) are normally trans-
ferred into the uterus, while the earlier stages of embryos are
transferred to the oviduct. In rodents the early stages can be trans-
ferred to the ovarian bursa.

The ready availability of hamster embryos, in contrast to more
expensive animals, make this species an attractive model in which to
study the normal development of preimplantation embryos. The tech-
niques used in this study for freezing preimplantation embryos were

based on reports by Whittingham gt_gl, (l972) and Tsunoda gt al,

(l976). The goals of the present study were the following: l) to
develop a simple, repeatable and objective method for hamster embryo
freezing and transfer; 2) to evaluate tests of frozen-thawed hamster
embryos that correlate with their viability; 3) to define the optimal
conditions for low temperature preservation and thawing of hamster
embryos; and 4) to examine the effect of various factors on implanta-
tion of unfrozen embryos and these factors include: a) developmental
stages of preimplantation embryos: b) recovery times of natural vs.
superovulated embryos; c) superovulation vs. natural ovulation;

d) intrabursal vs. intraoviductal transfer; e) unilateral vs. bilateral
transfer; f) left horn vs. right horn; 9) PBS vs. TC-l99 medium;

h) young (14 wk) vs. mature (28 wk) hamster; i) number of transferred

embryos per transfer; 3) embryo migration.

REVIEW OF LITERATURE

A. Embryo Freezing

 

l. Freezing Principles

In general, freezing causes death to most living organisms.
Some biochemical reactions can be slowed or stopped by freezing,
while others can be accelerated. Freezing is commonly used for
preservation of cells and tissues. Normally, freezing leads to ice
formation in the external medium and supercooling of the cell to -lO°C
or -15°C. This means that the membrane of the cell protects the
supercooled interior of the cell against the growth of external ice.
The intracellular vapor pressure is higher than the external ice
because of the supercooled water that occurs. This high vapor pressure
causes the cell to lose water in order to reach equilibrium with the
external ice. The cell undergoes dehydration, concentrates the solutes
and this leads to low intracellular aqueous vapor pressure. The events
that occur during cooling primarily depend on the velocity of cooling
and on the cell permeability (Mazur, 1970).

The critical cooling rate which produces internal ice
depends on the ratio of the voume of the cell to its surface area
and on its permeability to water. Large spherical cells are less
permeable to water and should have a lower critical cooling rate than
smaller more permeable cells. Exposure of the cell to temperatures
below 0°C leads to supercooling. The cooling rate and cell per-
meability to water are two important factors which aid the cell in

regaining equilibrium. Cells with a high permeability to water or

3

which are slowly cooled are equilibrated by the transfer of intra-
cellular water to external ice, i.e., they equilibrate by dehydration.
Conversely, cells with a low permeability to water or which are cooled
rapidly equilibrate partly by intracellular freezing. Small crystals
are produced which are enlarged during warming because of their high
surface free energies. Freezing causes dehydration or intracellular
crystal formation and both of these lead to loss of water and an
increase in the intracellular and extracellular solute concentrations.
Bank and Maurer (1974) suggested that the high survival of
frozen rabbit embryos was due to slow cooling, which permitted suf-
ficient time for the embryos to equilibrate osmatically with the
concentrated extracellular solutions. They found that the embryos
were not sensitive to differences in the warming rates between l.3°C
and 25°C/minute and that survival decreased at warming rates above
25°C per minute. They noted that the thermal and the osmotic factors
were important in determining rabbit embryo survival after freezing.
Maurer and Haseman (1976) indicated that the optimal condi-
tions for freezing and thawing rabbit morulae were not the same as
for 8-cell embryos. They also found that 2.8M or greater DMSO concen-
trations gave a low survival after thawing. This may be due to de-
hydration of the cells (Meryman, 1974) or to the toxic effect of high
DMSO concentrations produced during freezing (Meryman, 1971).
Whittingham (l977a) stated that the embryos of different species
have different sensitivity to cooling depending on the stage of
development. This was confirmed by Polge (l977) who found that cells
were more sensitive to slow warming than to rapid warming when rapidly

cooled. This was due to formation of ice crystals within the cells.

5

Leibo (1977) described the events that occurred during cooling and
thawing. Lower temperatures result in ice crystal growth inside

the cell leading to an increased concentration of dissolved solutes.
Warming melts the ice and exposes the cell to solute dilution.
Further, slow cooling to below the freezing point causes first, ice
formation at a certain point below freezing and second, the quantity
of ice nucleation increases to reach a chemical potential equilibrium
for that subzero temperature. Third, the amount of ice formation
increases in response to lowering the temperature of the solution
causing increasingly concentrated solutes. These progressive changes
continue as the temperature is lowered. Leibo (1977) stated that the
osmotic responses of the mouse embryos are time and temperature de-
pendent and all respond osmotically to maintain chemical potential
equilibrium across their membranes. Leibo (1977) found that the total
cell volume that an ovum contains when it freezes intracellularly is
substantially less at lower than at high cooling rates. The occurrence
of intracellular ice and the reduction in cell volume coincide with
each other at the same ranges of cooling rates. Ova cooled at rates
of 1°C/min or slower decrease in volume during cooling and few of them
freeze intracellularly.

Mazur (1963) presented equations for the osmotic response
of the cell to the increasing solute concentration produced by
progressive freezing. The change in cell volume rate depends on the
cell's surface area, the cell's original volumes the cell's perme-
ability to water and finally the temperature coefficient of that
permeability. By this model it is possible to measure intracellular

ice formation during freezing. Leibo (1977) stated that this model

6
can approximately describe the actual response of mouse ova to freezing
and also indicated that the response of ova and embryos to freezing is
not unique. Whittingham (l977b) found that the reasons for the
difference observed between species and developmental stages in the
sensitivity of embryos to cooling to 0°C were unclear. Results from
mouse studies have demonstrated that fast cooling rates produce ice
formation intracellularly, which is incompatible with the survival of
embryos when thawed slowly. Gradual lowering of temperature will
allow enough time for the cell to gain osmotic equilibrium with the
extracellular solution by progressive loss of water (Leibo and Mazur,
1978). In contrast, rapid cooling will not give sufficient time for
the cell to remain in osmotic equilibrium, and, as the temperature is
lowered, the cell contents will become increasingly supercooled.
This causes the water to freeze intracellularly, which leads to death
of the cell. Usually prior to freezing the embryo is mixed with
dimethyl sulfoxide (DMSO) or glycerol. This protects the embryo against
intracellular ice formation and gives a higher survival rate. The
permeation of the solutes are temperature and concentration dependent.
The greater the concentration and the higher the temperature of ex-
posure, the faster the solute permeates the cell membrane. These
cryoprotectants protect the cell from freezing damage by reducing the
electrolyte concentrations by virtue of their colligative action
(Mazur, 1979). They are usually present in molar concentration and,
when the cells are cooled slowly with addition of one of the cryopro-

tectants, cell survival normally will be high.

2. Freezing of Preimplantation Embryos
a. Mggjg_

The primary requirement of the freezing medium is for
the maintenance of embryonic viability during embryo collection,
freezing, transfer and manipulation in vitro (Whittingham, 1979). The
most suitable types of media are simple physiological salt solutions
buffered with phosphate or Hepes and supplemented with glucose, pyru-
vate, bovine serum albumin or bovine serum and antibiotics (Whittingham,
1971b, 1979). These media normally have stable pH in air (7.2 - 7.4)
and are superior to bicarbonate-buffered media previously used for
most embryo transfers where the pH cannot be maintained in air alone
(Whittingham, 1979). High pH and wide and rapid temperature fluctua-
tions are detrimental to hamster embryo survival (Whittingham and
Bavister, 1974). Phosphate and Hepes-buffered media are suitable for
low temperature storage (-l96°C) and also support the developmental
stage of 8-cell embryos and morulae for at least two cleavage divisions
at 37°C (Betteridge, 1977). Whittingham and Wales (1969) found that the
addition of glycerol or DMSO to Ringer's medium at 4 to 5°C for 24 hours
of storage had no effect on embryo survival. The return of embryos to
normal size was more rapid with DMSO than with glycerol, perhaps due
to the rapid movement of DMSO through the cell membrane causing
a normal osmotic equilibration. All preimplantation stages of mouse
embryos survived freezing and thawing best with Dulbecco's phosphate-
buffered saline (PBS) supplemented with pyruvate, glucose, bovine serum
albumin and antibiotics (Whittingham, gt_al,, 1972; Wilmut, 1972;

Whittingham, 1974). The same medium was used for 4- and 8-cell rabbit

8

embryos and morulae (Bank and Maurer, 1973; Whittingham and Adams,
1974), morulae and blastocysts of sheep (Willadsen gt_g1, 1976), and
cattle blastocysts (Wilmut and Rowson, 1973; Trounson et_al,, 1978).

Dulbecco's PBS supplemented with 0.1% glucose and 0.3%
to 1.0% BSA or 10% heat-inactivated fetal calf serum was used as a
freezing medium for zona-free hamster ova (Fleming gt_al,, 1979). Ova
were stored at -50°C for 3 and 120 days. Of the 152 frozen ova 84%
showed normal morphological appearance upon thawing. Quinn gt_§1, (1982)
observed a significant difference in the penetrability of frozen-thawed
hamster oocytes when comparing PBS and Hepes-buffered Tyrode's medium.
Hamster oocytes frozen in Hepes-buffered Tyrode's medium survived much
better (76%) when slow cooling was terminated at -80°C instead of -40°C
(66%) before transfer to liquid nitrogen. In contrast, mouse embryos
frozen in PBS showed no difference in survival whether slow cooling was
terminated at -40°C or -80°C (Whittingham, gt_al,, 1979). High
penetration percentages observed in frozen hamster ova when Hepes-
buffered medium containing 4 mg BSA/m1 was used as a freezing medium
(Quinn__t._l., 1982). Optimal survival rates of frozen hamster oocytes
were obtained with PBS medium containing high protein (10% fetal calf
serum)concentration (Fleming gt_al,, 1979). The higher survival rates
of hamster oocytes stored in HT6 freezing medium rather than PBS could
be due to the difference in protein concentration and buffering system
(Quinn, _t._l,, 1982). Phosphate buffered medium is detrimental to
survival of mouse embryos at 37°C (Quinn and Wales, 1973).

Whittingham gt_al, (1979) observed the highest survival

levels of 8-cell mouse embryos (88%) after transfer to liquid nitrogen

from -40°C, while with blastocysts 74% survival was obtained. The

highest survival rate (76%) was obtained when DMSO in PBS was diluted
from the sample at 37°C and added either at 0°C or 37°C before freezing
(Whittingham, 1974). Trounson §t_al, (1978) used PBS supplemented

with 20% fetal calf serum and found that 52% of the frozen-thawed cow

blastocysts continued normal development jn_vitro and 40% resulted in

 

pregnancies when transferred to recipients. When PBS medium was sup-
plemented with 50% rabbit serum, only 48% of the morulae appeared
normal. Thirty-one morulae were transferred to six recipients and this
resulted in higher pregnancy (100%) and implantation (39%) rates
(Tsunoda gt_al,, 1982). These observations indicate that there are
other important factors besides the freezing medium that can affect

directly embryo viability.

b. Seeding Temperature

 

Ice formation (seeding) in the freezing medium is
usually induced either by touching the side of the freezing ampule
with forceps precooled in liquid nitrogen or by adding crystals of
frozen medium. The effect of seeding on embryo survival and the op-
timum temperature for seeding has not been explored extensively (Moore
and Bilton, 1977). Usually when the embryo is either not seeded or
seeded below the optimal seeding temperature, then the freezing medium
will not freeze spontaneously. This results in supercooling and in-
adequate dehydration with an increased intracellular freezing (ice
formation inside the cell) upon reaching the nucleation temperature.
This is incompatible with the survival of the embryos, causing serious
intracellular damage (Leibo, 1977; Leibo and Mazur, 1978).

Whittingham (1978) observed that supercooling of 8-cell embryos

10

below -6°C markedly reduced embryo survival. Sheep morulae and blasto-
cysts demonstrated high sensitivity to seeding temperature. No
embryos developed in culture after seeding at -10°C or when not
seeded while 30 to 50% of sheep embryos developed after seeding at
-2.5, -5.0 or -7.5°C (Moore and Bilton, 1977). It is obvious that

a certain amount of intracellular ice is compatible with the survival
of mammalian embryos, since slowly cooled embryos when transferred
directly from the freezing apparatus at -30°C and -40°C to liquid
nitrogen survived only if rapidly thawed (Willadsen, 1977; Whitting-
ham, 1978). These results clearly indicate the necessity for seeding
at temperatures above -lO°C.

Miyamoto and Ishibashi (1981a) studied the effect of
seeding temperature on survival of frozen-thawed mouse morulae. They
found that seeding at -4 to -8°C had no effect on the survival rate in
the presence of 1.2 M ethylene glycol or 1 M glycerol. However, seeding
at -lO°C or below significantly reduced survival rates of frozen-thawed
morulae. The developmental rate, in terms of live young, was markedly
decreased when seeding was induced at -13°C than -4°C. These data
indicate that the seeding temperature is critical for embryo survival
and that seeding should be induced at a temperature slightly below the

freezing point of the cryoprotectant.

c. Freezing and Thawing Rates

 

Above a critical cooling rate survival of cell decreases
with increasing freezing rate (Mazur, 1970; Leibo and Mazur, 1971).
This is a manifestation of cell injury due to intracellular ice forma-
tion as evidenced by electron microscopy (Walter gt 31., 1975; Leibo,

1977). Whittingham et, a1, (1972) obtained high survival of mouse

11

preimplantation embryos (80%) after slow cooling (0.3 - 0.4°C/min)

and slow warming (4 - 25°C/min). Very few embryos survived either
after freezing at rates faster than 2°C/min or rapidly thawing the
embryos after slow cooling at O.3°C/min. When mammalian embryos are
cooled rapidly they become more sensitive to slow thawing. This is due
to the damaging effect of slow thawing, possibly by the growth of ice
crystals within the embryos (Polge, 1977).

Leibo gt 31. (1974) showed that the survival of frozen
8-ce11 mouse embryos depends as much on warming rate as on cooling rate.
Embryos cooled at l.7°C/min survived when thawed at rates from 1 to
100°C/min, whereas embryos slowly cooled at 0.18°C/min or 0.2°C/min,
required warming rate of 2°C/min to give 90% survival. If warmed
at 200°C/min very few survived (Leibo, et_gl,, 1974; Leibo, 1977).

This means that in the frozen state embryos cooled at 0.2°C/min differ
from embryos cooled at 2°C/min, indicating that during the freezing
process changes occur in the embryos that only manifest themselves
during thawing. Kasai gt El: (1980) observed that when mouse morulae
were cooled rapidly at 17°C/min and thawed rapidly at 360°C/min a
higher percentage survived than if thawed slowly at 25°C/min. Following
rapid freezing and thawing 70 to 82% of these embryos developed to
fully expanded blastocysts. Willadsen gt_al, (1976) and Willadsen
(1977) showed that a freezing rate of 0.3°/min and thawing rate of
lO°C/min in medium containing 1.5M-DMSO resulted in 70% survival of
late sheep morulae and early blastocysts. No embryos survived when
the freezing rate was increased to 1°C/min. Different cooling and
thawing rates were found to be necessary for 7- and 12-day cow

blastocysts. None of the advanced blastocysts survived when the same

12

freezing and thawing rates of early blastocysts were applied to the
freezing of 12-day old blastocysts. Higher survival rates of 12-day
old blastocysts (advanced) in 2.5 M-DMSO medium were obtained when
freezing rate increased to 1.2 - 2.4°C/min (Trounson, 1977).

The temperature at which intracellular ice occurs (nu-
cleation temperature) depends on both the extent of cellular shrinkage
and the composition of the solution within the cell. Freezing condi-
tions in which cellular shrinkage is not complete allow intracellular
ice to form and this correlates with cell death (Farrant, gt_al,,
1977). The damage to the mammalian cells on freezing can be due to
osmotic stress across cell membranes during or after thawing (Bank,
1973; Farrant, 1977). Damage to the embryos in the absence of cryo-
protectant appears directly related to concentration of the electro-
lytes, whereas injury in the presence of the cryoprotectants seems
related to volume changes (Mazur, 1977). Membranes at lower tempera-
tures are less resistant to deformations produced by osmotic changes
in the cell volume. The survival of mammalian embryos slowly frozen
in a cryoprotectant can also depend on the thawing rate. Rapid
thawing is more detrimental to embryo survival than slow thawing
(Whittingham, gt_al,, 1972; Wilmut, 1972; Leibo, gt_al,, 1974). When
8-cell mouse embryos are cooled rapidly, 70% or more survived if they
had first been cooled slowly to -50°C, but none survive if they are
slowly cooled to only -30° (Leibo, gt_§1,, 1974). This is due to
intracellular ice nucleation which occurs at -45°C (Leibo, gt_gl,,

1974; Leibo, 1977).

13

3. Fertility of Frozen Embryos

The temperature at which embryos are held ig_vjtrg_is a very
important factor in determining the viability of mammalian embryos.
Alliston gt_al, (1965) reported an increase in post-implantation em-
bryonic mortality after 1- and 2-ce11 rabbit embryos were maintained
at 40°C for 6 h ig_vjtrg, Staples (1967) cultured day 5 rabbit blasto-
cycst for 8, l6 and 24 h at 37°C and upon transfer to foster mother
found that 40%, 28% and 0% developed respectively. Table 1 shows the
viability of mammalian embryos after storage at temperatures between
0°C and 10°C. Chang (1947) demonstrated that rabbit embryos at the
2-cell stage were damaged by cooling to 5 or 0°C in less than 4 h.
However, Mauer (1962) showed that 8-cell rabbit embryos and early moru-
lae placed directly into medium at 0°C and cooled at 3°C/min or
0.02°C/min (stored for 2 days) gave viable young upon transfer. Mouse
embryos seem to tolerate cold shock and show less susceptibility to
changes in storage temperature (Whittingham and Wales, 1969).

Whittingham, et_gl, (1972) and Wilmut (1972) showed that
frozen mouse embryos from 1-ce11 up to the blastocyst stage are capable
of normal development upon thawing and transfer to foster mothers.
Sixty-five percent of the recipients became pregnant and 40% of the em-
bryos in the pregnant mice resulted in normal offspring. The 1-ce11
and 2-cell embryos were less sensitive to freezing and thawing than
blastocysts. Similar results were obtained by Wilmut (1972) with frozen

mouse blastocysts. The frozen blastocysts were tested by an jfl_vitro

 

culture technique and 80% survival was observed. Similar freezing
regimens have been used for freezing rat, rabbit, sheep, goat and cow

embryos (Whittingham, 1979). Table 2 shows the fertility of frozen

14

embryos. Whittingham and Bavister (1974) studied the development of

fertilized hamster ova after jg_vitro culture and transfer to foster

 

mothers. No viable young were obtained from these transfers. Experi-
ments on freezing hamster oocytes showed that 87 to 94% of the frozen
oocytes appeared normal after thawing. However, when these ova were

fertilized in vitro, very high percentage showed polyspermy (Tsunoda,

 

_t_al,, 1976). Quin gt_al, (1982) found that the rate of decondensa-
tion of human spermatozoa was faster in freshly collected oocytes

than in frozen-thawed ova. Parkening and Chang (1977) found that
there was a significant difference in the fertilization rate of
frozen-thawed ova from immature and mature mice, but this was not true
in the rat. Rabbit embryos are more sensitive to osmotic changes
during freezing and thawing. Dilution of the 1.6 M-DMSO at 37°C was
necessary to obtain high survivals. Sixty-five percent of the frozen-
thawed 8-ce11 embryos developed to blastocysts in culture, and subse-
quent transfer to suitable recipients resulted in 15% newborn (Bank
and Maurer, 1973, 1974).

Cow and pig embryos show a high sensitivity to cooling temper-
atures above 0°C. Eight-cell cow embryos demonstrated the highest sen-
sitivity to freezing (Wilmut gt_al,, 1975). When cow blastocysts were
thawed rapidly after slow cooling in 1.5 M-DMSO and following embryo
transfer to recipients, two newborn calves were born (Wilmut and Rowson,
1973). Very low resistance was observed in pig embryos to cooling. When
these were cooled to 0°C, none of them survived following thawing, and
very few embryos survived below 15°C. Upon transfer to recipients,
none resulted in pregnancy (Polge §t_al,, 1974; Polge, 1977). The

critical temperature which causes damage to pig embryos seems to be

15

 

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17

around 15°C, since a11 embryos survive above this temperature and the
damage at 15°C may be associated with 1ipid phase changes within the

membranes (Po1ge, 1977).

B. Embryo Transfer

 

1. Natura1 and Superovu1ation for Embryo Production

Superovu1ation regimens have been used wide1y for induction of
1arger numbers of embryo from superior donors. This has important eco-
nomic benefits especia11y in the 1ivestock industry (Foote and Onuma,
1970; Betteridge, 1977; Saumande and Pe11etier, 1975). Superovu1ation
responses in anima1$ are dependent on severa1 factors inc1uding breed,
nutrition, popu1ation sizes of ovarian fo11ic1es, seasona1 variations
and hormona1 preparation used.

Preimp1antation embryos are usua11y obtained from the fema1e
reproductive tract fo11owing natura1 or induced ovu1ation. In 1ater
case gonadotropins (PMS and LH or HCG) are used for maximum embryo
recovery. Embryo co11ection is performed in sma11 rodents by f1ushing
the oviduct and uterus, whereas in primates, rabbits and 1arge animaTS,
1aparotomy or Taparoscopy can be used. Advanced embryonic stages in
the cow and mare are recovered by non-surgica1 procedures but this is
not possib1e in other farm anima15 due to the anatomica1 nature of the
fema1e reproductive tract (Betteridge, 1977; Brackett, 1978; Whitting-
ham, 1979). The timing of ovu1ation by superovu1ation regimen varies
according to the species (Whittingham, 1979). Several variab1es affect
the recovery rates. These inc1ude: 1) dose and interva1 of gonado-
tropin, 2) route of injection, 3) stage of estrous cyc1e and 4) age

of animaT (F1eming and Yanagimachi, 1980; Mizoguchi and DukeTow, 1981).

18

Greenwald (1962) reported that injection of 30 iu PMS on day l of the
estrous cycle of the hamster resulted in the ovulation of an average

of 70 eggs. Golden hamsters superovulated by injection 5 to 25 iu PMS
followed 44 to 48 h later with similar doses of HCG resulted in good re-
covery rates (Tsunoda, gt_gl,, l976). Low recovery rates were observed
(mean of l5 to 20 ova per hamster) in superovulated hamsters with a
higher dose (40 iu) of PMS and HCG (Moore and Bedford, l978). An
average of 57 ova per hamster were recovered with 30 iu PMS followed
with 30 iu of HCG 72-76 h later (Mizoguchi and Dukelow, 1981).

Fleming and Yanagimachi (l980) found that the minimum interval between
PMS and HCG to obtain 83% ovulation and an average of 36 ova per hamster
ovulated was 44 h. Mizoguchi and Dukelow (198l) reported that super-
ovulation significantly increased the number of degenerated oocytes in
aged hamsters.

The developmental potential of embryos from superovulated and
naturally ovulated animals is similar, since normal offspring were ob-
tained from superovulated embryos. Experiments with hamsters (Fleming
and Yanagimachi, 1980), mice (Smith and Chrisman, l975) and rabbits
(Maurer, gt_gl,, 1968), confirmed the normality of newborn animals
resulting from superovulation. 0n the other hand, a higher incidence
of chromosomal abnormality has been reported in superovulated preimplan-
tation embryos in the mouse (Maudlin and Fraser, 1977), hamster
(Mizoguchi and Dukelow, l981) and rabbit (Fujimoto, gt al,, l974).
Takagi and Sasaki (l976) observed that superovulated embryos were un-
able to extrude the second polar body and they believed that the meiotic
maturation of the oocyte might have been affected by superovulation.

High polyspermic fertilization was observed in superovulated mouse

19

oocytes by Maudlin and Fraser (1977) and they suggested that the
cortical reaction which prevents polyspermy is defective in super-

ovulated eggs at least jg_vitro. High doses of PMS (30 iu) were

 

found to induce unilateral pregnancies in hamsters. This effect is
probably due to the alteration in the rate of gamete's or embryo's

transport to the uterus (Fleming and Yanagimachi, 1980).

2. Site of Deposition and Type of Transfer
The transfer of preimplantation hamster embryos has

been studied by several investigators (Blaha, 1964; Orsini and
Psychoyos, 1965; Sato and Yanagimachi, 1972; Ghosh §t_al, l982).
Hamster embryos (from 1-cell to blastocyst stages) can be trans-
ferred to synchronous recipient females by three different routes:
intrabursal (I.B.), intraoviductal (1.0.) and intrauterine (I.U.)
transfer. The site of embryo deposition selected is dependent on the
developmental stage of the embryo (Sato and Yanagimachi, 1972; Ghosh
§t_al,, 1982). The early preimplantation embryos from l-cell to 8-cell
stages are usually transferred I.0. to synchronous recipients (Whitting-
ham, 1979). Tarkowski (1959) obtained successful 1.0. transfer of all
preimplantation stages when these embryos were transferred to recipient
mice on the first day of pregnancy or pseudopregnancy. However, there
is species variation in the ability of early preimplantation embryos to
continue normal development in the recipient uterus. This usually de-
pends on the developmental stage of the embryos and the uterine environ—
ment of the recipient (Whittingham, 1979). Noyes gt El: (1963) found
that the intrauterine transfer of l- and 2—cell embryos in rats and

mice resulted in degeneration of these embryos within 24 h following the

20

transfer, but Moore and Shelton (1964) reported that the early preim-
plantation embryos (from 2- to 8-cells) survived to term when deposited
in the uterus of recipient sheep. Higher survival was obtained
however, by the 1.0. transfer of the same embryos. In mice and rats
higher survival of transferred embryos is obtained with recipients

that mated one day after the donors (Whittingham, 1979).

Moore and Bedford (1978) developed a new technique for transfer of
hamster oocytes by depositing the ova in the ovarian bursa (intra-
bursal transfer) using a fine micropipette. They found that the major-
ity of these ova passed into the oviduct within 15 min. A higher per-
centage of in_vjvg fertilization rate (96%) was observed with cumulus-
oophorus covered ova transferred by this method. The majority of these
eggs were found to be fertilized within 4 h when transferred at the
time of ovulation. This technique was utilized for studying the effect
of drugs on ovulation (Martin et_gl,, 1981). Orsini and Psychoyos
(1965) reported 62% implantation when hamster blastocysts were trans-
ferred by I.U. route into progesterone-treated ovariectomized re-
cipients. Sato and Yanagimachi (1972) were unable to get implantation
of l- and 2-cell hamster embryos transferred 1.0. However, they ob-
tained (80%) implantation in synchronized hamsters when donor blasto-
cysts were transferred to the uterus. Of these, 70% developed normally.
Higher survival rates were observed in mice when bilateral transfer was
performed rather than unilateral transfer (Muller and Carter, 1973).
Similar results were obtained in rabbits when frozen-thawed morulae
were transferred bilaterally (48%) compared to unilateral transfer

(23%) (Tsunoda gt_al,, 1982).

21

3. Transfer Media

 

The chemical composition, pH, storage period and the tempera-
ture of the transfer media are important variables that affect embryo
survival (Maurer, 1976; Betteridge, 1977, 1981). Simple medium such
as PBS with pH of 7.4 and supplemented with protein (usually 20% donor's
serum), an energy source (glucose, pyruvate, lactate) and antibiotics
maintain embryo viability in culture and support the subsequent embry-
onic development upon transfer (Betteridge, 1977). However, these
media are unable to support all the developmental stages of the preim-
plantation embryos. Whittingham (1975b) was able to obtain complete
preimplantation development in the mouse and also Maurer (1978) in
rabbit when bicarbonate-buffered media was used as culture media.

Hamster and mouse blastocysts transferred in TC-199 and
Whitten's media resulted in healthy newborn (Sato and Yanagimachi, 1972;
Moler _t_al,, 1979). Fifty percent implantation was reported in
rabbits when PBS medium supplemented with 5% fetal calf serum was used
as the transfer medium for rabbit blastocysts (Rottmann and Lampeter,
1981). Moler _t_al, (1979) used Whitten's medium at 37°C for non-
surgical embryo transfer in mice. The transfer of 278 blastocysts to
30 recipients resulted in a 90% pregnancy rate and 45% live young.
Table 3 shows the effect of transfer media on survival of unfrozen
mammalian species at temperatures between 25°C and 37°C.

The early preimplantation embryos of hamster, rat and

guinea pig showed higher sensitivity to culture media and jg_vitro

manipulation. This means they cannot tolerate jg_vitro culture com-

 

pared to late stages of embryonic developement (Sato and Yanagimachi,

1972; Whittingham and Bavister, 1974; Whittingham, l978).

22

TABLE 3

Viability of Mammalian Preimplantation Embryos
in Various Media at Temperature between 25°C and 37°C

 

 

Animals Developmental Transfer Percent References
Stage of Embryos Medium Viable
Young
Mice 2-cell Chemically 31 Whitten & Biggers
defined medium (1968)
Mice Morulae Synthetic 37 Hoppe & Pitts
medium (1973)
Mice Blastocysts Whitten's 45 Moler et a1.
medium (1979)
Hamster Blastocysts TC-199 56 Sato & Yanagamachi
(1972)
Rat 8-cell Brinster's 24 Maurer (1976)
medium
Rabbit 4-ce11 Bovine and 50 Maurer §t_al.
rabbit serum (1970)—'
Rabbit Blastocysts Modified F10 40 Staples (1967)
with serum
Sheep Morulae Sheep serum 16 Moor & Cragle

or TC-199

(1971)

 

23

4. Age of Recipient

 

The degree of synchronization between donor and recipient is
one of the most important factors for successful embryo transfer (Sato
and Yanagimachi, 1972; Adams, 1973; Fukumitsu and Sugie, 1974;
Betteridge, 1977; Whittingham, 1979). In small rodents implantation
takes place within a short period of time after the entrance of the em-
bryo to the uterus, therefore successful transfer depends on synchronous
embryo transfer. Asynchronous transfer in rodents is successful only
when the recipient females are mated one day later than the donors.
This type of synchronization is useful for embryos that show develop-
mental delay during handling in vitro (Whittingham, 1979). Sato and
Yanagimachi (1972) demonstrated that older hamster embryos (4-day
blastocysts) survived better after embryo transfer than younger embryos
(3-day embryos, 4- or 8-cell embryos). The transplantation of 8-cell
embryos from aged donor hamsters to young recipients resulted in a
49% survival rate. However, only 8% of embryos of the same stage
developed into fetuses when transferred to older recipients (Blaha,
1964). Although the transfer of hamster blastocysts to ovariectomized
progesterone-treated (artificially delayed implantation) recipients
resulted in live fetuses, lower rates of fetal development were ob-
served when the reproductive stages of the donor and recipient animals
were asynchronous (Orsini and Psychoyos, 1965). A high pregnancy rate
(100%) was observed in synchronized recipient hamsters receiving
blastocysts compared to 4- and 8-cell embryos (50% and 87% respectively,
(Sato and Yanagimachi, l972). Ghosh et_al, (1982) found no significant
difference in implantation rates of 2-cell and 8-cell embryos in

mature golden hamster (61% vs 64% respectively).

24

A low implantation rate was reported with 8-cell mouse
embryos from donors 2.5 days postcoitus, transferred to recipient
3.5 days postcoitus, and the removal of the zona pellucida did not
increase the implantation rate (McLaren, 1969). Toyada and Chang (1974)
reported that of 203 2-cell embryos fertilized jn_vjtrg_and transferred
to oviducts of 2-day pseudopregnant rats, only 21% showed fetal develop-
ment. Adams (1973) studied the effect of asynchronization between
donors and recipients on rabbit embryo survival. He showed that the
age of the transferred embryos was important for the maintenance of the
corpus lutea, which became critical around 14 days postcoitus. Embryos
up to 3 1/2 days younger than the corpus lutea were luteotrophically
competent whereas those 5 1/2 days younger were not. Whittingham and
Adams (1976) attributed the low survival of frozen-thawed rabbit
embryos after transfer to damage to the zona pellucida during freezing
or unfavorable synchronization between embryos and recipients. In
general the fertility of preimplantation embryos decreases with the in-
creasing age of donors and recipients. Many workers observed a marked
decrease in embryonic development, implantation rate, litter size and
fecundity in older hamsters (Soderwall et 21,, 1960; Connors, 1969,

1972; Parkening and Soderwall, 1973).

5. Number of Embryos Transferred

 

Sato and Yanagimachi (1972) transferred 3 to 5 hamster
embryos (4-, 8-cell and blastocysts) to each uterine horn. Of 206
embryos 63 percent implanted and of these 73 percent developed into
fetuses. The transfer of 78 rat embryos (7 to 8 embryos per transfer)

1.0. resulted in 42% implantation, 27% fetuses and 25% viable young

25

(Toyoda and Chang, 1974). The preimplantation and postimplantation
losses of the embryos in this study were 57% and 15% respectively.

They reported that rat ova fertilized jn_vjtrg_are capable of normal
development when transferred at the 2-cell stage. Table 4 shows the
development of mammalian embryos and the effect of the number of embryos
transferred on development. Maurer gt_al, (1970) found that rabbit em-
bryos transferred (2 to 5 per oviduct) after 62 h in culture gave 80
percent pregnancy rate compared to 89% pregnancy rate with direct trans-
fer. The period of pseudopregnancy in the recipient rabbit is an
important factor that affects the transferred embryos. When 5 to 10
morulae were transferred to each uterine horn in a 3 to 7 day pseudo—
pregnant rabbit, it resulted in 78% pregnancy and 39% newborn. Only
18.7% became pregnant and 1.7% gave newborn when embryos were trans-
ferred to 9-11 days pseudopregnant rabbits (Adam, 1971).

Studies in pigs showed that the transfer of large number of embryos
to recipient oviducts is not a major factor that limits litter size.
Recipients receiving 24 embryos per oviduct have higher pregnancy rate
compared to recipients receiving 12 embryos at 25 days of gestation.

The 24-embryo recipient gilts were found to have from 14 to 23 normal

embryos per litter at slaughter (Pope §t_al,, 1972).

6. Embryo Migration

 

There are no reports in the literature on this subject in ro-
dents. However, embryo migration is well documented in the pig (Anderson
and Parker, 1976; Pope gt_al,, 1982 a, b), ewe (Scanlon, 1972) and cow
(Rowson gt al,, 1971; Scanlon, 1972). Pope gt_§l, (l982a) studied

the relationship between myometrial contractility and intrauterine

265

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27

embryo migration in pigs. They found that myometrial contractility
increased with embryo migration. In_yjtrg_studies showed that day 12
pregnant horn flushings overcame significantly the inhibitory effects
of indomethacin on myometrial contraction. They found a short-acting
substance in the pregnant horn flushings that mimicked the stimulatory
effect of embryo on myometrial contraction. It was concluded that the
factor responsible for uterine contraction was hormonal in nature (Pope
_t_al,, 1982a). The popular hypothesis for the mechanism of embryo mi-
gration is the peristaltic contraction of the myometrium that facili-
tates migration of the embryos (Boving, 1971; Pusey gt_al,, 1980; Pope
_t_al,, 1982b). It was demonstrated that estradiol-178 and histamine
are involved in intrauterine migration of porcine embryo. When silastic
beads were impregnated with cholesterol or estradiol (E2) after 5 days
in the uterus, the beads which were impregnated with E2 migrated sig-

nificantly farther than those impregnated with cholesterol (Pope gt_gl,,

1982b).

MATERIALS AND METHODS

A. Animals

Healthy mature cycling golden hamsters (Mesocricetus auratus)

 

three to six months of age (Charles River Laboratories, Wilmington,
Mass.) were housed under a controlled l4 h:10 h (lightzdark) cycle
(light 0600 to 2000). Animals showing at least three consecutive

4-day estrus cycles were used.

B. Embryo Recovery and Handling

 

Donor females were housed with fertile males for 12 h from 1900.
Mating was confirmed by lordosis response and vaginal aspiration and
examination for sperm the following morning. The donors were killed by
cervical dislocation 26 to 31 h later (to recover l-cell embryos), 35
to 43 h (to recover 2-cell embryos), 52 to 69 h (to recover 4-cell
embryos), 68 to 71 h (to recover 8-cell embryos) or 77 h (to recover
morulae) after mating. Embryos were flushed from the oviducts and
uterine horns with a 30 ga. needle using either TC-199 medium (GIBCO,
Grand Island, NE) or Dulbecco's Phosphate Buffered Saline (PBS, GIBCO
Laboratories, Grand Island, NE). Hamster preimplantation embryos from
l-cell to the morula-stages were recovered as shown in figure 1 either

from the oviduct or uterine horn.

C. Freezing Techniques

 

The preimplantation embryos were washed twice with fresh medium
following recovery. They were placed in an ampule containing 0.1 ml

of the freezing medium (TC-199 or PBS) at 0°C. Next, 0.1 ml of

28

29

Hours after HCG injection i.P

 

 

 

 

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I I I I I I
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Hours after mating

Figure l. Embryo recovery times from golden hamster

30

3 M dimethylsulphoxide (DMSO) was added at 0°C. The final concentration
of DMSO in the ampule was 1.5M. The ampule was sealed and placed in a 95%
ethanol bath at 0°C and then seeded from -5°C to -10°C. Seeding was
induced by immersing a forceps in liquid nitrogen and touching the
ampule. The seeded embryos were cooled at a rate of 0.33°C/min (from
0.30 - 0.36°C/min) and when the final temperature of the ampule reached
-85°C (from -80° to -90°C) the ampule was plunged into liquid nitrogen
at -l96°C and stored from 1 to 90 days.

The freezing system which was used in this study consisted of an
unsilvered Dewar flask of six Torr vaccuum. Three hundred ml of 95%
ethanol was placed in the Dewar flask. The Dewar flask was then placed
inside a larger silvered flask which contained liquid nitrogen. The
depth of the Dewar flask was controlled by a laboratory elevator jack.
The temperature of the seeding ampule was recorded at 15 minute
intervals. The freezing rate was calculated as a function of tempera-
ture and time.

Slow thawing was performed by placing the frozen ampule in a 600
ml pyrex beaker containing 300 ml of 95% cold ethanol at -125°C. The
ampule was allowed to thaw slowly at a rate of l.5°C/min. Rapid
thawing was performed by direct transfer of the frozen ampule from the
liquid nitrogen into an ice-bath (0°C). This gave a thawing rate of
90°C/min. As soon as the ampule thawed the DMSO was diluted by step-
wise addition of 0.2, 0.2 and 0.4 ml of the medium at 30 second inter-
vals and finally 1 ml of the medium. The ampule was washed again
with 1 ml of medium. The washed medium was placed in a watch glass
and examined for embryo recovery under a dissecting microscope. The

thawed embryos were examined for morphological normality and trypan

31

blue exclusion (0.2%TpB in PBS). The tested embryos were washed twice

with fresh medium and prepared for embryo transfer.

0. Embryo Transfer

 

1. Basic Technigue for Transfer

 

Recipient hamsters were anesthetized with 0.12 ml penta-
barbitol-sodium (60 mg/ml) intraperitoneally. Embryos were transferred
to the ovarian bursa (I.B.), oviduct (1.0.) or uterus (I.U.) of the
recipients by making a dorsolateral incision through the abdominal wall.
The embryos from 1- to 8-cell were transferred through fine micro-
pipette into ovarian bursa or oviduct. The 4-cell, 8—cell and the
morulae were injected into the uterine horn through a micropipette or
a 26 gauge needle connected to PE-20 intramedic polyethylene tubing
(Clay Adams, Becton, Dickinson and Company, Parsippany, NJ). The
other end of polyethylene tube was joined to a one m1 tuberculin syringe
or mouth piece. The embryos were also transferred to the uterine horn
through a fine micropipette connected to a plastic mouth tube. The
micropipette and the polyethylene tube were checked after each embryo
deposition to assure that all embryos were transferred. The recipients
were killed on day six or fourteen following the transfer and examined

for implantation sites and embryonic development.

2. Superovulation and Recipient Synchronization

 

The day of postovulatory vaginal discharge was designated as
day l of the cycle. Females were superovulated with an intraperitoneal
injection of PMS (Sertropin, Teizo Company, Ltd., Tokyo, Japan).
Twenty-five IU/100 gm of body weight at 1000 on any day of the

32

4-day cycle, followed 76 h later by an intraperitoneal injection of HCG
(APL, Ayerst Laboratories, Montreal, Canada), 25 IU/100 gm of body
weight at 1400 . Mating was as described earlier. The period of
synchronization was the same for the donor and recipient groups. Re-
cipients were ear marked, shaved, anesthetized and prepared for embryo

transfer.

3. Age of Animals

 

The donor and recipient groups were both superovulated and
synchronized. According to the age of donor and recipient, the animals
were divided into two groups, namely young 14 wk old (12 to 16 wk) and
mature 26 wk old (24 to 28 wk). The effect of age of animals on

implantation rates was studied.

4. Number of Embryos Transferred

The effect of the number of embryos per each transfer on the
implantation rate and embryo viability was examined. Recipients were
divided into three subgroups depending on the number of transferred
embryos, and each subgroup received 3-6, 7-9 and 10-12 embryos per
transfer respectively.

Chi-square, Bonferroni chi-square and T-test were used for
statistical analysis of the data (Gill, 1978). If the level of signifi-

cance was not stated otherwise in the table the p>.05 was used.

5. Embryo Migration
To study the migration of early preimplantation embryos, all

the surgical transfers were made unilaterally either to left or right

ovarian bursa, oviduct or uterine horn. The recipients were killed on

 

33

day 6 or 14 of pregnancy and implantation sites were checked in both
horns of the uterus. Migration of embryos to either left or right horn
was counted and the interaction between the developmental stages and

migratory capability of the embryos was also noticed.

RESULTS

1. Media_

The viability of various preimplantation embryos was not affected
by freezing medium TC-l99 (Table 5). The viability of l-cell and 2-cell
compared to morulae were significantly affected by PBS (p<.01) when
used as a freezing medium (Table 6). The developmental stages of the
embryos showed different responses (p<.001) to PBS medium (Table 6). The
fertility of frozen-thawed embryos was determined by the embryo trans-
fer technique. The fertility of these embryos was not affected by the
medium (Tables 7 and 8). The survival of hamster embryos in TC-l99 in
terms of normal and TpB' embryos was significantly higher (p<.01 and

p<.001 respectively) than in PBS medium (Table 9).

2. Seeding Temperature

 

Hamster preimplantation embryos were seeded at a temperature
range from -5°C to -23°C. The effect of seeding temperatures on
embryo survival was studied (Figures 2,3,4, 5 and 6). The optimal
seeding temperature for l-cell, 2-cell, 4-cell, 8-cell and morula was
shown to be -5, -7, -9, -9 and -5 respectively. The survival of pre-
implantation embryos in terms of embryo normality, TpB' and implanta—
tion rate was lowered when the seeding temperature was below -10°C.
Seeding temperatures between -5°C and -10°C did not have a deleterious
effect on hamster preimplantation embryos (Figures 2 to 6). The numbers
of normal, 193' and implanted embryos were significantly (p<;01)

increased when the embryos were seeded at temperatures above 10°C.

34

TABLE 5

*Viability of Frozen-Thawed Hamster
Preimplantation Embryos in TC-l99

 

 

Embryo Stage Number Number Number Number Number
Frozen Thawed Normal TpB'(%) Donors
(%) (%)
l-cell 149 114 (77) 100 (88) 106 (93) 10
2-cell 102 88 (86) 72 (82) 81 (92) 8
4-cell 42 33 (79) 27 (82) 28 (85) 4
8-cell 105 90 (86) 76 (84) 77 (86) 9
Morula 50 35 (70) 28 (80) 28 (80) 4
Total 448 360 (80) 303 (84) 320 (89) 35

 

* p>.05

36

TABLE 6

Viability of Frozen-Thawed Hamster Preimplantation Embryos

in Dulbecco's Phosphate Buffer Saline (PBS)

 

 

 

Embryo Number Number Number Number Number
Stage Frozen Thawed Normal TpB'(%) Donors
W W»)

1-cell 357 264 (74) 1925(73) 194 (73) 24
2-ce11 94 68 (72) 46a(68) 52 (76) 7
4-ce11 43 37 (86) 32 (86) 32 (86) 5
8-ce11 91 53 (58) 41 (77) 44 (83) 8
Morula 164 133 (81) 116 (87) 102 (77) 13
Total 749 555 (74) 427b(77) 424 (76) 57

a = p<.01 Significantly different from morula

b = p<.001 Morphologically normal embryos vs.

developmental stages

37

TABLE 7

*Fertility of Frozen-Thawed Hamster Embryos in TC-l99

 

 

Embryo Number Number Number Number
Stage Transferred Implanted Recipient Pregnant
(as) m
1-cell 34 25 (74) 5 4 (80)
2-cell 36 28 (78) 4 4 (100)
4-cell 20 16 (80) 3 3 (100)
8-ce11 42 35 (83) 8 8 (100)
Morula l7 14 (82) 3 3 (100)
Total 149 118 (79) 23 22 (96)

 

* p >.05

38

TABLE 8

*Fertility of Frozen-Thawed Hamster Embryos in PBS

 

 

Embryo Number Number Number Number
Stage Transferred Implanted Recipient Pregnant
W (%1
l-cell 88 66 (75) 15 13 (86)
2—cell 25 20 (80) 5 5 (100)
4-ce11 10 8 (80) 2 2 (100)
8-ce11 24 21 (87) 4 4 (100)
Morula 48 35 (73) 9 8 (88)
Total 195 150 (77) 35 32 (91)

 

*

p>.05

39

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mmm cw cusp gmcmw; zpucmugmwcmwm po.vq

 

 

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m u4m<k

40

100
.\ ONE-CELL EMBRYOS
\\ ’\
\\ .._. fps NEGATIVE
3 ‘\ r-41MPLANTATION
m
560 ‘\
> \‘
V\
\3
.. 'I
z “
m 1
U \
M
III-l
a. \
20 {\x
\“x
\ ‘\\\
-5 -10 -15 -2o

SEEDING TEMP. 'C

Figure 2. Survival of l-cell hamster embryos seeded at temperatures
between -5°C and -23°C. Each point represents 2 - 3 replicates.

p <.001: seeding temperature -5 to -10°C vs. -11 to ~15°C; p <.005:
implantation significantly different from morphological test and
trypan blue reaction at seeding temperature -11 to -15°C.

41

" ' TWO _ceu EMBRYOS
1
\l
80 {—x HMORPH. NORMAL
\ 9" TPB NEGATIVE
\ \\ o—--0IMPI.ANTAT[ON
\
2: 45C) \ \
no \ b\
< \ \
s I \
\\ \
4O \ \
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E" \ \
W K \
a. 20 \\ \
\ \
“- \\
\\\ \x
‘4
\~\
’ 5 '10 '15 '20
SEEDING TEMP. °C
Figure 3.

Survival of 2-cell hamster embryos seeded at temperatures
between -7 and -23°C. Each point represents 2-3 replicates. p <.001:
seeding temperature -7 to -10°C vs. -11 to -15°C; implantation sig-

nificantly different (p <.005) from morphological test and trypan blue
reaction at seeding temperature -11 to -15°C.

42

100 FOUR-CELI. EMBRYOS

 

so H MORPH. NORMAL
g—a TPB NEGATIVE
.3 o---o IMPLANTATION
S. 60
:>
’2' 4o )0
“I I
C) I
a: I
u: l
“ 1
12(3 I
l
l
\ l
-5 -10 -15 -2o -25
SEEDING TEMP. °c
Figure 4.

Survival of 4-cell hamster embryos seeded at temperatures
between -6°C and -20°C. Each point represents 2-3 replicates. p <.001:
seeding temperature -6 to -10°C vs. -11 to -15°C; implantation sig-

nficantly different (p <.005) from morphological test and trypan blue
reaction at seeding temperature -11 to -15°C.

43

10° 8 -CELL EMBRYOS

\ H MORPH.NORMAL
‘ .._. TPB NEGATIVE

a. ‘x g-.. IMPLANTATION
-' '3
9° \
$60 ‘ \\
> \‘ \
\
I— 1 \
:5 l \
IAI ¢ \
u \ \
as x \
n. \ \
I‘ \\ \\
12¢) \ \\
\ \
I \ \
\\ \
\ \
' 5 '10 '15 -20

SEEDING TEMP. 0(:

Figure 5. Survival of 8-cell hamster embryos seeded at temperatures
between -7°C and -l9°C. Each point represents 2-3 replicates.

p <.001: seeding temperature -7°C to -10°C vs. -ll°C to -l5°C; im—
plantation significantly different (p <.005) from morphological test
and trypan blue reaction at seeding temperatures -11 to -15°C.

VIABLE

PERCENT

44

100 MORULAE

o———.MORPH. NORMAL

7"», h—«TPB NEGATIVE
" .--,IMI>LANTATI0N

ON
C)

20

 

'5 -10 -15 -2o
SEEDING TEMP. “C

Figure 6. Survival of hamster morulae seeded at temperatures between
-5°C and -15°C. Each point represents 2-3 replicates, (p >.05).

45

Morulae tolerated seeding temperatures up to -15°C without apparent loss
in survival rate (Figure 6). The survivability of 1-, 2-, 4- and 8-cell
embryos seeded at a temperature range of -5°C to -23°C (Figures 2, 3, 4
and 5 were similar. The normality and TpB' of morula were similar
between seeding temperatures of -5 and -15°C. The number of implanted
morulae decreased when the embryos were seeded below -15°C (Figure 6).
The three tests of embryo viability were positively correlated (Figures

7, 8 and 9). The correlation coefficient between these tests was 0.99.

3. Freezing and Thawing Rates

 

There was no evidence (p>.05) that developmental stages of hamster
embryos were adversely affected by slow cooling (0.33°C/min) and thawing
(l.5°C/min) rates (Table 10). 0f the 576 thawed embryos, 81% were
normal morphologically and 84% by TpB'. The viability of preimplanta-
tion embryos in terms of embryo normality and TpB' was significantly
(p<.001) affected when these embryos were thawed at a rate of 90°C/min
(Table 11). This thawing rate (90°C/min) did not affect significantly
(p>.05) morulae and 8-ce11 embryos (Table 11). There was significant
interaction between morulae and l-, 2-, 4-cell embryos and also between
4-cell and 8-cell embryos (p<.05, Table 11) with respect to their
normality and trypan blue reaction. These developmental stages showed
higher sensitivity to rapid thawing (p< .001). The fertility of frozen-
thawed embryos was not adversely affected by either slow or rapid thawing
(p>.05, Tables 12 and 13). The viability of slow-thawed embryos was
significantly higher (p<.01) than that of those rapidly thawed (Table 14).
The number of pregnant recipients was not markedly different (p>.05)

between slow and rapidly thawed embryos. The developmental potential

ICND

6C)

TBP' EBRYOS

2C)

Figure 7.

normal embryos.

 

46

100

2t) 6!)
MORPH . NORMAL EMBRYOS

Positive correlation between TpB- and morphologically
Each point represents 3-4 replicates.

47

80
g = -99
Y : 2-87 '1' ° 73X
2
2 60
p—
S
2
S 40
G.
.75
20
l
20 4O 60 80
MORPH. NORMAL EMBRYOS
Figure 8.

Positive correlation between morphologically normal embryos
and implantation. Each point represents 3-4 replicates.

48

80 r = .99 ‘***9
Ay : 2.94 + '74x it“
I:
g 60
S
I!
‘I
z 40
E
72¢)

 

20 4o 60 80
TBP' EMBRYOS

Figure 9. Positive correlation between TpB' embryos and implantation
Each point represents 3-4 replicates.

49

TABLE 10

*Viability of Frozen Hamster Embryos Following Slow Thawing l.5°C/min

 

 

Cell Stage Number Number Number Number Number

Frozen Thawed Normal TpB'(%) Donors
1-CE11 217 171 (79) 141 (82) 152 (89) 15
2-cell 196 156 (79) 118 (76) 133 (85) 14
4-ce11 114 4(74) 69 (82) 69 (82) 11
8-cell 101 (6 2) 50 (79) 52 (83) 10
Morula 123 102 (83) 86 (84) 79 (77) 12
Total 751 576 (77) 464 (81) 485 (84) 62

 

*

p >.05

50

TABLE 11

Viability of Frozen Hamster Embryos Following Rapid Thawing 90°C/min

 

 

 

Cell Stage Number Number Number Number Number
Frozen Thawed Normal TpB (%) Donors
(%) (%)

1-ce11 212 140 (66) 94 (67)a 90 (64)b 14
2-ce11 228 193 (85) 124 (64)a 143 (74)a 15
4-ce11 69 60 (87) 30 (50)b 32 (53)b 6
8-cell 71 46 (65) 36 (78)c 37 (80)C 7
Moru1a 99 57 (58) 49 (86) 53 (93) 9
Total 679 496 (73) 333 (67)d 355 (72)d 51

a = p <.05 Significantly different from morula

b = p <.01 Significantly different from morula

c = p <.05 Significantly different from 4-ce11

d = p <.001 Over all data

51

TABLE 12

*Fertility of Slowly Thawed Hamster Embryos Following Embryo Transfer

 

 

Cell Stage Number Number Number Number
Transferred Implanted Recipients Pregnant
(%) (%)
1-cell 46 34 (74) 6 5 (83)
2-cell 61 48 (79) 8 7 (87)
4-cell 42 34 (81) 8 7 (87)
8-cell 24 21 (87) 5 5 (100)
Morula 40 29 (73) 7 6 (85)
Total 213 166 (78) 34 30 (88)

 

* p >.05

52

TABLE 13

*Fertility of Rapidly Thawed Hamster Embryos Following Embryo Transfer

 

 

Cell Stage Number Number Number Number
Transferred Implanted Recipients Pregnant
(%) (%)
l-cell 42 33 (78) 6 5
2-cell 58 39 (67) 6 4
4—cell 16 ll (69) 3 2
8-cell 22 18 (82) 4 4
Morula 28 16 (57) 4 3
Total 166 117 (70) 23 18 (78)

 

* p >.05

 

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moxLaEm coepmucmeasemgm memsm: cmNogu mo zueeepgmu

up mgm<h

54

TABLE 15

The Developmental Potential of Slowly Cooled and Thawed Hamster Embryos
in Either PBS or TC-199 Containing l.5M-DMSO

 

 

 

Embryo Number Number Number fetuses/ Number fetuses/
Stage Transferred Implanted* Number implanted** Number transferred
(%) (%) (%)
a,b a,b
l-cell 50 37 (74) 8 (22) (16)
2-cell 90 7o (78) 29 a”(41) a”(32)
4-cell 28 23 (82) 16 (70) (57)
8-cell 49 41 (84) 30 (73) (61)
Morula 35 28 (80) 22 (78) (63)
Total 252 199 (79) 105 d(53) d(41)
a = p <.01 Significantly different from morula
b = p <.01 Significantly different from 4- and 8-cell
embryos
c = p <.01 Significantly different from 8-cell embryos
d = p <.001 Total data: Developmental stages of the

embryos vs. developed fetuses

* Day 6 of pregnancy
** Day 14 of pregnancy

55

of slowly cooled and thawed hamster preimplantation embryos on day 14
of pregnancy was significantly (p<.001) affected by the developmental
stages of the embryo (Table 15). Small numbers of 1-cell and 2-ce11
embryos were developed to day 14 fetuses compared to 4-, 8-cell and

morula (Table 15).

B. Embryo Transfer

 

The transfer of 893 unfrozen embryos from 204 donors to 216
recipients resulted in 60% implantation (Table 16). There were no sig-
nificant differences between developmental stages and implantation
rates. The pregnancy rate of unfrozen embryos was higher for all
transferred preimplantation embryos (87%, Table 17). No significant
interactions were observed between developmental stages of the embryos
and pregnancy rates. The recovery times of superovulated embryos were
longer (9 h delay) than for naturally ovulated embryos (p<.001, Table 18).
There were significant differences between the recovery times and the
developmental stages of the embryos (p<.05). The implantation rates
were significantly lower (p< .005) in the superovulated group compared
to natural ovulation (Table 19). Higher implantation rates were observed
in naturally ovulated l-cell embryos (71%) compared to superovulated
l-cell embryos (54%, p<.05). The transfer of l-cell or 2-cell embryos
either I.B. or 1.0. gave similar results (Table 20). The intrauterine
transfer of 4-cell and 8-cell embryos was more effective (p<.05) than
the intrabursal transfer of the same developmental stages of the embryo
(Table 21). Bilateral transfer of preimplantation embryos showed lower
implantation rates (55%) than that of unilateral transfer (62%. p<.l).

The developmental stages of the embryos were not significantly affected

56

TABLE 16

*Implantation Following Unfrozen Embryo Transfer in Golden Hamster

 

Cell Stage Number Number Number Number Number Implan.

 

Exp. Donor Recip. Transf. Implan. Percent
l-cell 13 52 57 298 164 55
2-cell 23 102 103 343 211 61
4-cell 7 18 24 77 46 64
8-ce11 9 32 32 175 112 60
Total 52 204 216 893 533 60

 

* p >.05

57

TABLE 17

*Pregnancy Percentage in Golden Hamster
Following Embryo Transfer

 

 

Cell Stage No. Transf. No. Recip. No. Pregn. Pregn. %
Embryos Recip.

1-cell 298 57 48 84

2-cell 343 103 95 92

4-ce11 77 24 19 79

8-cell 175 32 26 81

Total 893 216 188 87

 

* p >.05

58

TABLE 18

Recovery Times of Hamster Embryos

 

 

Cell Stage Natura1 Ovulation Superovulation
Hour after mating Hour after mating
Mean (Range) Mean (Range)
l-cell a26 (24-37) 31 (18-50)
2-cell a36 (25.5-53) 43.14 (24-64)
4-cell a51.51 (Bl-53.5) 69.6 (65-74)
8-cell a68.7 (64-76) 72.52 (68-76)
Total b45.5 (24-76) 54.06 (18-76)

 

0)
II

p <.05 Significantly different from corresponding
superovulated embryos

0'
II

p <.001 Overall data

59

TABLE 19

Survival of Superovulated Hamster Embryos Following Embryo Transfer

 

 

Cell Stage Natural Ovulation Superovu1ation*
No. implanted/No. transferred No. implanted/No. transferred
(%) (%)
1-cell 30/42 a(71) 147/273 (54)
2-cell 57/85 (67) 154/258 (60)
4-cell 26/39 (67) 20/38 (53)
8-cell 37/55 (67) 75/120 (62)
Total 167/246 b(68) 396/689 (57)

 

* Both groups (donor and recipient) were superovulated

D.)
II

p <.05 Significantly different from super-
ovulated l-cell embryos

U'
11

p <.005 Overall data

60

TABLE 20

*Implantation in Golden Hamster Following Intrabursal and
Intraoviductal Transfer of Preimplantation Embryos

 

 

Cell Stage Intrabursal Transfer Intraoviductal Transfer
No. Implanted/No. Transferred No. Implanted/ No. Transferred
(%) (%)
l-cell 152/277 (55) 23/41 (56)
2-cell 184/299 (61) 27/44 (61)
Total 336/576 (58) 50/85 (59)

 

* p >.05

61

TABLE 21

Implantation in Golden Hamster Following
Intrabursal and Intrauterine Transfer

 

 

Cell Stage Intrabursal Transfer Intrauterine Transfer
No. implanted/No. transferred No. implanted/No. transferred
(%) (%)
4-cell 21/39 (54) 25/38 (66)
8-cell 25/47 (53) 87/128 (68)
Total 46/86 (53) 112/166 a(67)

 

a = p <.05 Significantly different from intrabursal transfer

62

TABLE 22

Effect of Side of Embryo Transfer on
Implantation in Golden Hamster

 

 

 

Cell Stage Unilateral Transfer Bilateral Transfer
No. implanted/No. transferred No. implanted/No. transferred
(%) (%)

1-cell 121/206 a(59) 43/92 (47)
2-cell 123/195 (63) 88/148 (59)
4-cell 33/55 (60) 19/32 (59)
8-cell 81/121 (67) 31/54 (57)
Total 358/577 b(62) 181/326 (55)

a = p <.l Significantly different from Bilateral Transfer

b = p < 1 Overall data

63

TABLE 23

Effect of Uterine Horn on Implantation of
Golden Hamster Embryos

 

Cell Stage Right Horn Left Horn
No. implanted/No. transferred No. implanted/No. Transferred
(% %

 

1-ce11 84/166 (51) 80/132 a(61)
2-cell 107/84 (58) 104/159 (65)
4-ce11 29/46 (63) 17/31 (55)
8-cell 53/78 (68) 59/97 (61)
Total 273/474 (58) 260/419 (62)

 

a = p <.05 Significantly different from right horn

64

(p>.05) by the type of transfer (Table 22). The transfer of l-cell
embryos to the left horn yield higher implantation rates (p<.05) compared
to the right horn (Table 23). Both horns were equally effective for the
transfer of other embryo stages (Table 23). Two transfer media (PBS

and TC-199) were used for embryo transplantation. Both media were found
to be effective except in 8-cell embryos (p<.01) where the implantation
rate was higher in TC-l99 than PBS (Table 24). The implantation rate

of preimplantation embryos obtained either from young (14 wk) 0r mature
(26 wk) hamsters did not differ significantly (p>.05, Table 25). The
numbers of transferred embryos had no significant effect (p>.05) on

the implantation rates of the developmental stages of the embryos

(Table 26). The transfer of 7 to 9 embryos vs. 3 to 6 or 10 to 12
embryos to recipient females showed relatively higher implantation

rate than the other two groups (66% vs. 61% and 57% respectively).

Embryo migration was determined by unilateral transfer of pre-
implantation embryos into one uterine horn. The different develop-
mental stages showed significantly (p<.05) different migratory capability
(Table 27). One-cell embryos had a higher migration rate (p<.05) than
the two-cell embryos. Migration of l-cell embryos to the left uterine

horn was significantly higher (p< .05) than to the right uterine horn.

 

65

TABLE 24

Viability of Hamster Embryos in Two Transfer
Media (PBS vs. TC-199)

 

 

Embryo PBS TC-l99 Number
Stage No. Implanted/ No. Implanted/ Recipients
No. Transferred (%) No. Transferred (%)

1-cell 78/140 (56) 86/158 (54) 57
2-cell 79/133 (59) 132/210 (63) 103
4-cell 17/32 (53) 29/45 (64) 24
8-cell 41/77 (53) 71/98 a(72) 32
Total 215/382 (56) 318/511 (62) 216

 

a = p <.01 Significantly different from PBS medium

66

TABLE 25

Implantation of Hamster Embryos in Young and Mature
Recipients Following Embryo Transfer

 

 

 

Cell Stage Young Hamstera Mature Hamsterb

No. Implanted/ No. Implanted/
No. Transferred (%) No. Transferred (%)

l-cell 100/189 (53) 64/109 (59)

2-cell 101/170 (59) 110/173 (53)

4-cell 29/50 (58) 17/27 (63)

8-cell 69/111 (62) 43/64 (67)

Total 299/520 (57) 234/373 (63)

a = Age range 12-16 weeks, mean: 14 weeks, young group

b = Age range 24-28 weeks, mean: 26 weeks, mature group

67

 

 

 

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68

 

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mm m4m<h

DISCUSSION

The survival of frozen-thawed hamster preimplantation embryos was
highest in TC-l99 medium compared to PBS medium. The l- and 2-cell
embryos showed higher sensitivity to PBS compared to morulae, whereas
no such observation was noticed in TC-l99 medium. The low survival
rates of hamster embryos in PBS medium could be due to the adverse
effect of the pH. The optimum pH for hamster embryos was found to be
7.2 - 7.4 (Whittingham and Bavister, 1974; Whittingham, 1979). PBS
medium is detrimental to the survival of mouse embryos at 37°C (Quinn
and Wales, 1973). In the present experiments PBS was not supplemented
with an external energy source. Therefore, the lack of protein and glu-
cose in the PBS may also be responsible for the low survival rate.

The results of the present experiments agree with those of Fleming
_t_§1, (1979) and Quinn gt_al, (1982). The later investigators attri-
buted the low survival rates of hamster oocytes in PBS to the low pro-
tein concentrations and the buffering system. Tsunoda and Sugie (1977a)
reported that the addition of rabbit serum to PBS resulted in a sig-
nificant increase in the developmental rate of rabbit embryos and also
in the number of newborn compared to PBS alone. One-cell and two-cell
hamster embryos are highly affected by culture media (Sato and
Yanagimachi, 1972; Whittingham and Bavister, 1974; and Whittingham,
1978) and the results of the present study support this. The fertility
of frozen-thawed hamster embryos was similar in both media and this
indicates that the developmental stages of the hamster embryos were not
affected by the freezing media. Similar results were obtained with

mouse embryos when PBS was used as a freezing medium (Whittingham gt_

69

70

al,, 1972; Wilmut, 1972). The survival rates of early preimplantation
embryos (following freezing and thawing) were markedly decreased when
these embryos were seeded below -10°C, whereas the late embryonic stage
was unaffected even when seeded at -15°C. These results demonstrate that
the early preimplantation embryos are highly sensitive to seeding
temperature and this confirms similar observations with mice
(Whittingham, 1977a; Miyamoto and Ishibashi, 1981a) and sheep embryos
(Moore and Bilton, 1977). The higher tolerance of hamster morulae to
the seeding temperature probably reflects the ability of morulae to
undergo sufficient dehydration during freezing at the time when intra-
cellular freezing takes place. Rall gt_gl, (1983) observed that the
nucleation temperature was responsible for the drastic decrease in the
survival of mouse embryos. Supercooling of mouse embryos below -7°C
significantly decreased survival rate and when mouse morulae were
seeded below -10°C there was a lower survival rate (Whittingham, 1977a;
Miyamoto and Ishibashi, 1981a). These findings are inconsistent with
regard to the observations with hamster 8-ce11 and morulae in the present
study. This discrepancy could be due to species specificity to seeding
and also to the methods of seeding induction. The implantation rates of
l- to 8-cell stages were significantly different from the other tests at
seeding temperatures below -ll°C. This finding suggests that implantation
is not dependent or related to the other tests. The three tests of sur-
vival were positively correlated at seeding temperatures above -10°C.

The viability and fertility of various preimplantation embryos
relative to slow cooling and thawing were similar. This means that all
the preimplantation embryos tolerate equally slow freezing and thawing

by adequate dehydration. These results agree with those of Tsunoda

71

.et__l. (1976); Parkening and Chang (1977); and Quinn gt_gl, (1982)

who reported similar results with hamster oocytes following slow
cooling and thawing. Rapid thawing after slow freezing significantly
lowered the survival of 1- to 4-cell hamster embryos in the present
experiments. Similar results were reported for mouse (Whittingham gt_
a1,, 1972; Wilmut, 1972; Liebo _t_al,, 1974), sheep (Willadsen, 1976)
and cow (Wilmut and Rowson, 1973) embryos. The lower survival rates of
hamster embryos after rapid thawing confirm previous findings of

Leibo gt_g1, (1974 and 1977). These results also support other in-
vestigators who found that rapid thawing is more detrimental to embryo
survival than slow thawing (Whittingham gt_al,, 1972; Wilmut, 1972; Bank,
1973: Leibo gt_al,, 1974; Farrant, 1977). It seems that cell membranes
at lower temperatures are less resistant to deformation produced by os-
motic changes and rapid thawing may cause osmotic shock to hamster
embryos. The results of the present study support the earlier sug-
gestion of Bank (1973) and Farrant (1977) who recommended slow thawing
for slowly cooled embryos to avoid cell damages by osmotic shock.

The highest sensitivity to development to day 6 and 14 fetuses was
observed in l- and 2-cell hamster embryos. The pre- and post-implanta-
tion losses of these embryos were 24% and 68% respectively. These
results are in contrast to those of Whittingham gt_gl, (1972) who
found that 1- and 2-cell mouse embryos were less sensitive to freezing
damage than blastocysts. These differences may be due to species
sensitivity to freezing and this was reported by several workers
(Tsunoda gt_al,, 1976; Maurer, 1976; Parkening and Chang, 1977; and
Whittingham, 1978 and 1979). The developmental potential of early

hamster embryos following slow freezing and thawing agrees with those of

72

Tsunoda and Sugie (1977b) who found lower developmental rates in 2-cell
rabbit embryos after freezing. The marked reduction in the develop-
mental potential of early hamster embryos could be because these
embryos were not cultured jn_vitrg_after thawing but immediately
transferred to recipients. This finding confirms that of Whittingham
and Anderson (1976) and Whittingham (19776) who showed that immediate
transfer of mouse embryos after thawing without culturing resulted in
a significant decrease in survival rate. These workers also reported
that mouse embryos demonstrate developmental delay after freezing and
in order to resume normal embryonic development a restorative period
in culture is required.

The pregnancy and implantation rates of unfrozen hamster embryos
were similar, which indicates that there are no specific interactions
between the development stages of hamster embryos and their ability
to implant. The uterine environment seems to have no selective effect
on the various developmental stages of hamster embryos. Kusanagi (1981)
found that embryo transfer had no effect on the developmental capability
of mouse embryos. The results of the present experiments agree with
those of Ghosh gt 21, (1982). Similar results also were obtained in
mice following embryo transfer (Moler §t_al,, 1979). However, the
findings of the present experiments are in contrast to those of Sato
and Yanagimachi (1972) who reported lower embryonic development in
hamsters after embryo transfer. These differences may be due to modi-
fication of the transfer medium and techniques in the present study.

Superovulation regimens induced a 9-hour delay in ovulation and also
significantly lowered implantation rates compared to naturally ovu1ating

embryos. These observations confirm those of previous workers who

73

reported that superovulation produced side effects in rodents (Fujimoto
__t__l., 1974; Takagi and Sasaki, 1976; Maudlin and Fraser, 1977;
Mizoguchi and Dukelow, 1981). Similar treatment in hamsters induces
unilateral pregnancy and alteration in ovulation and embryo transport
(Fleming and Yanagimachi, 1980). The lower implantation rates in
superovulated embryos could be due to the effect of hormones on the
embryo itself (Katzberg and Hendrickx, 1966; Shaver and Carr, 1967;
Shaver, 1970; Fujimoto gt_al,, 1974; Takagi and Sasaki, 1976) and/or
the uterine environment of the superovulated recipient (Yang and Chang,
1968; Banik, 1975, Betteridge, 1977). Similar ovulation delay was also
reported in golden hamster embryos by Ghosh gt_al, (1982).

The I.B. and 1.0. transfer of l- and 2- cell embryos did not show
differences in implantation rates; however, the I.B. transfer of 4- and
8-ce11 hamster embryos resulted in a marked decrease in implantation
rate compared to 1.0. transfer. The lower implantation rate of I.B.
transfers could be due to the adverse environment of the ovarian bursa
which might cause a delay in embryo transport and subsequently result
in aged embryos. It has been shown that the normal site for 4—cell and
8-cell embryos is the oviduct or uterus (Sato and Yanagimachi, 1972;
Whittingham, 1979: Ghosh gt_gl,, l982). One-cell and 2-cell mouse
and rat embryos undergo degeneration when transferred to the uterus
(Noyes gt_gl,, 1963), whereas they develop normally after transfer to
the oviduct (Tarkowski, 1959; Whittingham, 1979).

The unilateral transfer of hamster embryos yielded higher implan-
tation rates than bilateral transfer. This may be due to the longer
surgical stress and recovery period with bilateral transfer than uni-

lateral transfer. McLaren (1970) showed that an empty uterine horn

74

does not exert a systemic or local luteolytic effect on the survival
of mouse eggs transferred to the other horn, whereas Adams (1962) re-
ported that in rabbits the type of transfer had no effect on egg sur-
vival after transfer. These observations are consistent with results
of the present experiment; however, Muller and Carter (1973) in mice,
and Tsunoda gt_gl, (1982) in rabbits, observed a higher survival rate
of embryos transferred bilaterally. This discrepancy may be due to
species and also variation in transfer technique.

The viability of preimplantation hamster embryos in transfer media
was similar except that 8-cell embryos showed a higher sensitivity to
PBS than TC-199 medium. The same explanations for PBS as a freezing
medium are valid for PBS as a transfer medium for hamster embryos.

It is well known that the various developmental stages of embryos
demonstrate different responses to the same medium or different media
(Whittingham and Bavister, 1974; Hanada and Chang, 1976; Whittingham,
1979). Hamster oocytes and embryos are found whether fertilized in_vjvg_
or jn_vjtrg to be highly sensitive to culture media, especially to the
pH (Hanada and Chang, 1976; Whittingham, 1979). It was indicated from
the results of the present experiments that equivalent age of the
donors and recipient has no effect on implantation rate of hamster
embryos following embryo transfer. The relative higher survival of
hamster preimplantation embryos following embryo transfer which was
observed in the mature group rather than the young group may reflect
the increased maturation in the 26-week-old hamster. These results
agree with those of Mizoguchi and Dukelow (1981) and Evans and Dukelow

(1982) who found an increase in fertility with older hamsters.

75

The various numbers of the transferred embryos did not show a sig-
nificant interaction with respect to their implantation in the recipient
hamsters. The lower implantation rate in the 10 - 12 transferred embryo
group may be due to the spacing phenomenon, which could act as a stress
factor leading to preimplantation mortality. These results are consis-
tent with those of Sato and Yanagimachi (1972) who obtained similar
implantation rates after deposition of 3 to 5 embryos per horn.

Similar implantation rates were observed in mice, rats and hamsters
after the deposition of 5 to 8 embryos (Humphrey, 1967; Toyada and
Chang, 1974; Fiser and Macpherson, 1982), and this confirms the
results of the present study.

A 10% embryo migration was observed in hamsters following the
transfer of 903 embryos unilaterally. The highest migration was
observed in l-cell embryos. The migration of preimplantation hamster
embryos may be due to production of estradiol by the embryos which
causes uterine contraction and increases embryo migration. This con-
clusion is supported by Pope gt_al, (1982a) who found in pigs that
uterine contraction increases with embryo migration and the factor
responsible for uterine contraction was hormona1 in nature. Ghosh
gt al. (1982) attributed the higher uptake of estradiol in the uterine
tissue following 8-cell embryo transfer due to the increased production
of estradiol by the 8-cell embryo. This supports the higher migratory
percentage of the 8-cell embryos than the 2-cell and 4-cell embryos in
the present study. The production of estradiol from hamster embryos
has been suggested by several investigators (Dickmann and Sengupta,
1974; Dickmann §t_gl,, 1976; and Sengupta, gt_gl,, 1981). Dickmann
and Sengupta (1974) reported that hamster embryos contain

76

AS-BB-hydroxysteroid dehydrogenase and l7-B-hydroxysteroid dehydrogenase,
which are both involved in biosynthesis of steroid hormones. Other
workers observed that estradiol-l7 directly affects the transport of
mouse embryos and also has synergistic action with progesterone on

embryo transport and development (Roblero and Garavagno, 1979). These
findings are consistent with the stimulatory effect of embryonic
estradiol on embryo migration in the hamster. Another factor which
might be responsible for the migration of hamster preimplantation

embryos is the spacing phenomenon since higher migration was observed
with the deposition of larger number of embryos (10 - 16 embryos) in

the recipient females.

SUMMARY AND CONCLUSIONS

The golden hamster (Mesocricetus auratus) was studied as a model

 

for embryo production, freezing and transfer. Hamster preimplantation
embryos (1-ce11 to morula) were recovered after either natura1 ovu1ation
or superovulation. Survival of hamster embryos in different freezing
media, seeding temperatures, freezing and thawing rates was evaluated
by morphological appearance, trypan blue exclusion and embryo transfer
techniques. The developmental potential of slowly cooled and thawed
embryos was also examined by transfer of these embryos into syn-
chronized recipient females. Pregnancy and implantation rates of
various developmental stages of unfrozen hamster embryos and their
interactions with different factors were determined. The following
conclusions were obtained from the results of these studies:

1. TC—199 was superior to PBS as a freezing medium. The
l- and 2- cell embryos were adversely affected by PBS medium.

2. The early preimplantation embryos are highly sensitive to
seeding temperature, whereas late embryos tolerate temperatures as low
as —15°C without apparent loss in viability. Optimal seeding temper-
ature for hamster embryos was found to be above -9°C.

3. Higher survival rates for hamster preimplantation embryos
were found with slow thawing (l.5°C/min) after slow cooling (0.33°C/min).

4. The developmental potential of hamster embryos showed sig-
nificant interaction with the developing stages of the embryos. The

early preimplantation embryos were more susceptible to freezing damage.

77

78

5. Superovulated embryos showed a 9 h delay in ovulation and
had lower implantation rates than naturally ovulated embryos.

6. One-cell and 2-cell embryos should be transferred 1.0. or
I.B. but other embryos should be deposited in the uterus. The site
of transfer was found to affect significantly the developmental stages
of the embryo.

7. No significant effects were observed on the survival of un-
frozen embryos when placed in uterine horns and by the side of transfer.

8. TC-l99 as transfer medium is superior to PBS.

9. The transfer of 3 to 9 embryos per side from mature hamsters
(24 to 28 weeks old) is recommended.

10. Embryo migration was found in the hamsters and l-cell embryos

showed the highest migratory capability.

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APPENDIX A

 

92

APPENDIX A

TABLE 29

*Viability of Frozen-Thawed Cow Eggs Following
Seeding at Different Temperatures

 

 

Number Number Number Number Period Seeding
Frozen Thawed Normal TpB Frozen Temperature
(%) (%) (%)
48 46 (96) 34 (74) 39 (85) 17h -5°C
41 34 (83) 29 (85) 27 (79) 2d -6°C
57 48 (84) 42 (88) 43 (90) 2d -7°C
92 89 (97) 85 (96) 85 (96) 17d -8°C
238 217 (91) 190 (88) 194 (89) l7h-17d All
Temperatures

 

* r = 0.99 Morphologically normal embryos and TpB' Embryos

Cooling rate = O.25°C/min
Thawing rate = O.50°C/min
Freezing medium = TC-199 and PBS

93

APPENDIX A

TABLE 28

*Effect of Freezing Medium on Viability of Cow Eggs

 

 

Number Number Number Number Period Freezing
Frozen Thawed Normal TpB Frozen Medium
(%) (%) (%)

66 57 (86) 53 (93) 55 (96) 4h-12d PBS
139 130 (93) 110 (85) 115 (88) l7h-l7d TC-l99
52 43 (83) 37 (86) 36 (84) 30h-l7d DMEM
257 230 (89) 200 (87) 206 (90) 4h-l7d All Media

 

r = .999 Morphologically normal embryos and TpB- embryos

Slow cooling rate O.25°C/min
Slow thawing rate O.50°C/min
Seeding temperature = -7°C
Cow follicular oocytes

APPENDIX B

10.

11.

12.

95

APPENDIX B
PUBLICATIONS BY THE AUTHOR

Effect of high fluoride intake on the reproductive organs of mice.
M.S. Thesis, Department of Physiology, College of Veterinary
Medicine, Baghdad University, June, 1976.

Reversability of high fluoride intake effect on mouse hemoglobin
concentration. Iraqi J. Vet. Med. 1:141-154, 1977.

Effect of high fluoride intake on haematological aspects of the
mouse. Quart. J. Exp. Physiology. 63:83-88, 1978.

Effect of high fluoride uptake on reproductive system of the
female mice. Iraqi J. Vet. Med. 4:118-131, 1980.

Ig_vitro uptake of estradiol and progesterone in the hamster
uterus and embryo relative to the time of embryo transfer and
stage of development. SSR Abstract 246, p. 154A/August 10-14,
1981, Corvalis, U.S.A.

Ig_vitro uptake of estradiol and progesterone in the hamster
uterus relative to the time of embryo transfer and stage of
development. Steroids. 40:133-138, 1982.

Embryo production, freezing and transfer in the golden hamster.
Abstract. Amer. Soc. Animal Science, Guelph, Ontario, Canada.
August 8-11, 1982.

Fertility of frozen-thawed hamster preimplantation embryos.
Abstract. Amer. Ass. Advanc. Sci., Detroit, Michigan, U.S.A.
May 26-31, 1983.

Preliminary studies of the effects of sodium fluoride on chicken
embryos. Egyptian J. of Animal Sci. (in press).

Studies on development of hamster embryos of early cleavage stages.
Biol. Reprod. (submitted)

Survival of cow follicular oocytes frozen at -l96°C. (in prepara-
tion)

Deep double freezing of cow oocytes and hamster eggs and embryos.
(in preparation)

APPENDIX C

Name:
Born:

Birthplace:

Formal Education:

Degrees Received:

Experience:

Membership in:

97

APPENDIX C
CURRICULUM VITAE

MUNDHIR TAIYEB RIDHA

July 16, 1949

Kanakin, Iraq

Kanakin High School

College of Veterinary Medicine
University of Baghdad

Baghdad, Iraq

Bachelor of Veterinary Medicine and Surgery
College of Veterinary Medicine
University of Baghdad, 1974
Master of Science

College of Veterinary Medicine
University of Baghdad, 1976

Assistant Lecturer and Laboratory Supervisor
in Physiology, Histology and Embryology

Research Scientist

University of Baghdad

Department of Physiology and Biochemistry
University of Basrah

Department of Biology and Chemistry
1977-1979

Society for the Study of Reproduction
American Society of Animal Science

American Association for the Advancement
of Science

International Embryo Transfer Society

Iraqi Veterinary Medical Association