/ . Embryo!, exp. Morph. Vol. 29, 1, pp. 133-144, 1973
Printed in Great Britain
133
The development of rabbit eggs
in the ligated oviduct and their viability after
re-transfer to recipient rabbits
By C.E.ADAMS 1
From the A.R.C. Unit of Reproductive Physiology and Biochemistry,
University of Cambridge
SUMMARY
The development of fertilized rabbit eggs trapped in the oviduct by ligation was examined
at 84, 96, 108 and 120 hp.c. in ten animals, five of which were superovulated. Out of 210 eggs
recovered, only two failed to develop into early blastocysts. The proportion of blastocysts
undergoing some expansion increased from 66% at 84 h to 100% from 96 h onwards, when
a high proportion, 66-89%, had ruptured zonae pellucidae. The viability of the blastocysts
was assessed by transfer to the uteri of recipient rabbits, in which the proportion developing to
term fell from 62% at 84 h to 3 % at 108 h.
A total of 643, 2- and 4-cell eggs recovered 30 hp.c. was transferred to the ligated oviducts
of oestrous, early or mid-luteal does for periods of 24, 48 or 72 h. Development to the early
blastocyst stage appeared to be faster in the luteal phase than in oestrous does. Of the eggs
recovered after either 48 h or 72 h, 20-40 % and 3-17 % respectively were capable of developing to term. In general, the less advanced blastocysts survived better.
Three hundred and twenty-one 60 h morulae were transferred to the ligated oviducts of
oestrous or mid-luteal does for 24,48 or 60 h. Neither the rate of egg development nor viability
was significantly affected by the endocrine status of the temporary recipient. The proportions
of eggs developing to term after transfer to recipients were 50%, 20% and 0%.
It is concluded that the rabbit egg cannot develop beyond the early blastocyst stage in the
oviduct, irrespective of the host's endocrine status. If development is interrupted, the life-span
of the blastocyst is very short. The rabbit blastocyst requires to undergo almost continuous
development to differentiate normally.
INTRODUCTION
In mammals, with certain exceptions which include the cat (Manwell &
Wickens, 1928), dog (Hoist & Phemister, 1971), mink (Hansson, 1947) and pig
(Pomeroy, 1955), the eggs enter the uterus about 3 days after ovulation, most commonly as morulae but sometimes as early blastocysts (see reviews by Boyd &
Hamilton, 1952; Blandau, 1969). If fertilized eggs are restricted to the oviduct by
ligation, they may develop to the early blastocyst stage in the mouse (Kirby,
1962; Orsini & McLaren, 1967; Weitlauf, 1971), rabbit (Pincus & Kirsch, 1936;
Adams, 1958), rat (Alden, 1942) and sheep (Wintenberger-Torres, 1956), but
apparently not in the pig where blastulation fails (Murray et al. 1971). Moreover,
1
Author's address: 307 Huntingdon Road, Cambridge CB3 OJQ, U.K.
134
C. E. ADAMS
the fertilized eggs of one species may survive and develop to the early blastocyst
stage in the oviduct of another, e.g. the rabbit can act as host to: cow (Adams,
Moor & Rowson, 1968; Lawson, Rowson & Adams, 1972), mouse (Brinster &
Thomson TenBroeck, 1969), pig (Polge, Adams & Baker, 1972), and sheep
(Lawson, Adams & Rowson, 1972). Presently, all available evidence suggests that
irrespective of whether the egg is confined to a native or alien oviducal environment development fails at the early blastocyst stage and degeneration then
occurs. Within species the loss of viability of tube-locked eggs has only been
investigated in the sheep (Wintenberger-Torres, 1956) and mouse (Weitlauf,
1971). In the rabbit, Adams (1958) observed that though egg development failed
at approximately 84 h p.c, there appeared to be some delay, estimated at 12 h,
before signs of impending degeneration appeared. However, no direct estimate
of the viability of such blastocysts is available, and since it is recognized that
morphological criteria may be poorly correlated with developmental potential,
the present work was undertaken. Another objective was to investigate the
development and viability of either 2- or 4-cell eggs or late morulae following
their transfer for limited periods to the oviducts of rabbits in different, welldefined endocrine states. The fate of 60 h morulae transferred to the oviduct on
the 11th day of pseudopregnancy has been described recently (Adams, 1971).
MATERIALS AND METHODS
The animals are conveniently divided into three main groups according
to treatment, consisting of (1) donors to provide fertilized eggs, (2) temporary
recipients to which eggs were transferred for periods of 24-72 h, and (3) recipients in which the further development of these eggs was assessed. Three
experiments were carried out. In Exp. 1 the native fertilized eggs were trapped
in the oviduct by ligation for varying lengths of time before recovery and transfer. In Exp. 2 and 3 eggs recovered either 30 or 60 hp.c. were transferred to the
oviducts of temporary recipients, which were either oestrous, early luteal or
mid-luteal. Altogether a total of 232 sexually mature does was used; 38 in
Exp. 1, including 10 donors and 28 recipients; 120 in Exp. 2, including 26 donors
and 25 temporary recipients and 69 recipients; and 74 in Exp. 3, including 16
donors, 12 temporary recipients and 46 recipients. The experimental animals
consisted mainly of a large breed (strain A) supplemented with Dutch belted
stock (Adams, 1910 a) derived from our own closed colonies.
Donors
All of the donors, except 5 in Exp. 1, were treated with a horse anterior
pituitary preparation to induce superovulation, as described previously (Adams,
1971). Twelve hours after the final priming injection the does were mated with
two fertile males and given 25 i.u. human chorionic gonadotrophin (Lutormone,
Burroughs Wellcome) to ensure the induction of ovulation.
Development and re-transfer of rabbit eggs
135
In Exp. 1 each of the donors' oviducts was ligated with cotton thread near the
tubo-uterine junction at either 14-24 h p.c. or 36-48 hp.c. to prevent the eggs
entering the uterus. Normally the rabbit egg enters the uterus between 72 and
80 hp.c. (Assheton, 1894; Gregory, 1930). Eggs were recovered from the donors
at autopsy by flushing 2 ml warm (37 °C) sterile 0-9 % NaCl solution through
the excised oviducts, at 84-120 h p.c. (Exp. 1), 30 h p.c. (Exp. 2) or 60 h p.c.
(Exp. 3). Using a zoom binocular microscope ( x 20 to x 80), the flushings were
then examined for eggs which were counted and quickly classified according to
stage of development and condition.
Temporary recipients and recipients
Does intended for use as temporary recipients were either left untreated
(oestrous), or mated with a vasectomized male and, during the autumn and
winter months, given an ovulating injection of HCG, either 24-30 h (early luteal)
or 9-11 days (mid-luteal) before egg transfer. Recipients were similarly treated
48-96 h before egg transfer, which was arranged so that the stage of egg development, rather than the eggs' absolute age, would be synchronous with the recipients'
luteal development. Thus, in Exp. 1 the recipients were 86—101 hp.c.; in Exp. 2
either 72 or 96 h p.c. for eggs that had spent 24-48 h or 72 h respectively in the
temporary recipients; and in Exp. 3 either 72 h or 96 h for eggs that had spent
24 h or 48-72 h respectively in the temporary recipients. In one exceptional
group (Exp. 2) involving re-transfer to the oviduct, the five recipients were
48 h p.c.
Laparotomy was performed under Nembutal (pentobarbitone sodium,
60 mg/ml Abbott Laboratories) and Fluothane (Halothane B.P., I.C.I. Ltd)
delivered in oxygen. In the case of the temporary recipients, eggs were transferred to the oviducts using a smooth-ended pipette which was inserted via the
fimbria to a depth of 3-4 cm before the fluid, about 0-02 ml, containing the
eggs was gently expelled. Each oviduct was ligated near the utero-tubal junction
with cotton thread. The subsequent recovery and examination of eggs from the
temporary recipients were as described above. Apart from the small group
already referred to, all re-transfers were to the uterine horns, using the technique
described by Adams (1962). The recipients were kept till full term in order to
record the number of young born. Pregnancy was diagnosed by palpation on the
11th day p.c.
RESULTS
Experiment 1: development of fertilized rabbit eggs trapped in the oviduct by
ligation
The results of restricting fertilized eggs to the oviduct, expressed in terms of
the stage of development attained and condition of the eggs at recovery, are
shown in Table 1. At 84 h only early blastocysts were recovered, including onethird unexpanded and two-thirds expanding but still intact. Within the next 12 h
the position changed significantly: thus, at 96 h all of the blastocysts had
136
C. E. ADAMS
Table 1. Stage of development and condition of eggs recovered from the ligated
oviducts of rabbits 84-120 h post coitum, and their survival after transfer to the
uteri of synchronized recipients
Stage of egg development
Eggs
recovered
from
ligated
oviduct
No.
(h p.c.) animals
84
96
108
120
3
4
2
1
A
,
v
Early blastocysts
A
/
Expanding
No.
eggs
41
76
65
28
Un-
A
,
Morulae expanded Intact
0
0
2
0
14
0
0
0
27
26
5
3
Proportion of eggs
developing to term (%)
K
N ,
s
Recipients
*
All
with
Ruptured recipients
0
50
58
25
62
35
3
0
implants
89
50
8
0
expanded and in two-thirds of them the zona pellucida had ruptured. Thereafter, the proportion of blastocysts with ruptured zonae pellucidae increased
still further, reaching 89 % at 108 h, after which, however, there was no further
change, except for progressive degeneration of the cellular component.
Practically all of these eggs were transferred to recipients. Of those recovered
at 84 h a high proportion was capable of developing to term (62 %), but by 96 h
viability had declined to 35 %, and it fell to almost zero during the next 12 h
period. The declining pregnancy rate noted among recipients of 108 h eggs
(3 out of 7 compared with 70 % at 84 and 96 h) indicates that an increasing
proportion of the older eggs was dying before or near the time of implantation.
Experiment 2: development of 2- and4-cell eggs in the ligated oviduct ofoestrous,
early luteal or mid-luteal recipients
Altogether 643 eggs were transplanted to 9 oestrous, 9 early luteal and 7 midluteal recipients for periods of 24,48 or 72 h. The results are presented in Table 2.
The proportion of eggs recovered varied from 74-5 to 96-9 %, with no consistent
difference existing between groups. Irrespective of the hosts' endocrine status,
all of the eggs recovered after 24 h were at the morula stage, containing approximately 16 cells, which is normal. At 48 h, however, development had proceeded
further in the luteal phase animals, which yielded 60% unexpanded early
blastocysts compared with only 21 % in the oestrous recipients where the
proportion of morulae was correspondingly greater. This slower rate of development was still apparent at 72 h as judged by the significantly higher proportion
of morulae, namely 38 % compared with 4 %. At this time the two early luteal
does yielded a high proportion of expanding early blastocysts (82 %) whereas
none of the blastocysts recovered from the mid-luteal phase doe had expanded.
This variation may reflect inherent differences between donors rather than an
effect of treatment.
137
Development and re-transfer of rabbit eggs
Table 2. The development of 30 h 2- and4-cell rabbit eggs transferred to the ligated
oviducts ofoestrous, 1-day, or 9- to 11-day pseudopregnant rabbits
Stage of egg development
A
Time eggs
No. eggs
Temporary recipient
spent in
A
r
> temporary f
Condition
No. recipient (h) Transferred Recovered Morulae
Oestrous
Early luteal
Mid-luteal
24
Oestrous
Early luteal
Mid-luteal
48
Oestrous
Early luteal
Mid-luteal
\
Unexpanded Expanding
65
[55
41
63
49
41
63
49
(142
85
[118
116
75
101
92
30
39
24
45
62
34
49
24
13
2
1
16
7
23
55
2
Early blastocysts
P6
72
27
5
40
0
Table 3. The survival of 30 h 2- and 4-cell rabbit eggs exposed for 1-3 days to the
ligated oviducts ofoestrous, 1 day or 9- to 11-day pseudopregnant recipients, and
then re-transferred to the uteri of synchronized recipients
(In parentheses the no. of recipients pregnant on day 11.)
Condition of temporary ;recipient
temporary
recipient
(h)
24
48
72
Early-luteal
Oestrous
Eggs
\
(
No.
eggs
re- Young born
No.
trans- ,
'*
v
No.
0/
recips.
recips. ferred No.
/o
5(3)
12(11)
4(2)
37
77
29
2
31
5
5
40
17
Mid-luteal
f
5(3)
5*0)
10(5)
6(2)
No.
No.
eggs
eggs
re- Young born
re- Young born
K
'\
•
trans- i
*
No. trans- i
0/
ferred No.
recips. ferred No.
/o
/o
31
31
69
36
3
3
16
1
10
10
23
3
5(1)
38
1
3
13(12)
4(4)
96
23
19
4
20
17
* Tubal transfer
Details of the various transfer groups are given in Table 3. No more than 10 %
of the morulae recovered after spending 24 h in the temporary recipients subsequently developed to term after transfer to the recipients' uteri and the results
were no better following transfer to the oviduct. The relatively low pregnancy
rate indicates that some pregnancies failed completely either before or about the
time of implantation. Of the eggs that spent either 48 h or 72 h in temporary
recipients, 20-40% and 3-17% respectively subsequently developed to term.
The lowest rate of survival involved the early luteal group which contained
138
C. E. ADAMS
Table 4. The development of60 h morulae transferred to the ligated oviducts of
oestrous or 9- to 11-day pseudopregnant rabbits, and their survival after transfer to
the uteri of synchronized recipients
Stage of egg development
Time eggs
Temporary
spent in
recipient
temporary
recipiCondition No.
ent (h)
Mid-luteal
Mid-luteal
Oestrous
Mid-luteal
1}
No. eggs
Early blastocysts No. young
A
A
,
,
* born/no.
(
TransReUnExeggs transferred covered Morulae expanded panding
ferred
24
/40
145
37
40
1
4
29
30
7
6
20/37
22/39
48
/98
192
83
71
3
15
22
18
58
38
16/80
13/71
60
|16
130
16
24
0
1
4
5
12
18
0/24
a significantly higher proportion of expanded early blastocysts than either the
'oestrous' or 'mid-luteal' groups. Once again the low pregnancy rate suggests
that the majority of the blastocysts succumbed very early, probably prior to
implantation.
Experiment 3: development of 60 h morulae in the ligated oviduct of oestrous and
mid-luteal recipients
A total of 321, 60 h morulae was transferred to the ligated oviducts of six
oestrous and six mid-luteal does, where they were allowed to remain for 24,
48 or 60 h. The results are presented in Table 4. The proportion of eggs recovered,
which varied from 77 to 100 %, appeared to be little affected either by the endocrine status of, or time spent in, the temporary recipients. Equally it appears
from Table 4 that egg development was relatively unaffected, except that its
rate was somewhat slower in the luteal than in the oestrous does. After 48 h, for
example, 21 % of the eggs transferred to luteal phase does were still morulae
compared with only 3-6% in the oestrous does, and there was a corresponding
difference in the proportion of expanding early blastocysts (53-7% versus
69-9 %). The development of the eggs recovered after 60 h was similar irrespective of the endocrine status of the temporary recipient.
Practically all of the eggs were re-transferred to synchronized recipients where
their viability proved to be almost identical, irrespective of previous treatment.
Of eggs that had spent 24 h in a temporary recipient more than 50 % developed
to term, but after a further 24 h only 20 % survived, with a decline to zero after
a further 12 h. The sharp decline in viability is reflected in the pregnancy rate,
which fell from 92 % (12/13) to 56 % (13/23) to 0 % (0/4) for the 24, 48 and 60 h
groups respectively. In the 48 h group a high proportion of the eggs must have
Development and re-transfer of rabbit eggs
139
died either before or near the time of implantation, and this applies particularly
to those from the oestrous temporary recipients.
Representative examples of eggs recovered from animals in Exp. 1, 2 and 3 are
shown in Fig. 1 (C-H), which also includes two normal early blastocyst stages
(A, B) for comparative purposes. The results relating to the viability of the eggs
shown in Tables 1, 3, 4 are depicted diagrammatically in Fig. 2.
DISCUSSION
The present results confirm earlier findings (Pincus & Kirsch, 1936;
Adams, 1958) that rabbit eggs fail to develop beyond the early blastocyst stage
when restricted to the oviduct. They also show that up to 96 hp.c. a substantial
proportion of such early blastocysts was capable of developing to term, but
that during the next 12 h viability was rapidly lost. A similar situation was found
to exist when 60 h morulae were cultured in vitro for 1-4 days (Adams, 19706).
In that case, too, blastocyst expansion was limited and rupture of the zona
pellucida was common. In the rabbit the action of a special uterine protein,
'blastokinin', whose appearance coincides with cavitation and blastocyst expansion has been invoked as an inducer and regulator of blastocyst development
(Krishnan & Daniel, 1967). More recently it has been proposed that blastokinin
'possibly acts at the transcriptional level in the role of a derepressor' (Gulyas &
Krishnan, 1971). In an extensive review of the biochemistry of oviducal secretions (Hamner & Fox, 1969) no mention was made of blastokinin having been
found in the rabbit oviduct, and recent work (Urzua, Stambaugh, Flickinger &
Mastroianni, 1970) indicates it is absent there. Feigelson & Kay (1972) consider
it improbable that a unique post albumin band, which they found in tubal fluid
of oestrous rabbits, is identical with blastokinin, though having similar electrophoretic properties.
In contrast to the fate of rabbit eggs transferred to the asynchronous uterus
(Adams, 1971), neither the rate of development of 60 h morulae in the ligated
oviduct nor their subsequent viability was significantly affected by the endocrine
status of the temporary recipient. Reference to Tables 1 and 4 shows that the
rate of development of the transferred eggs was slower than that of the native
eggs restricted to the oviduct; at 96 hp.c, for example, all native eggs had undergone expansion into early blastocysts, whereas even at 108 h(60 hmorulae + 48 h),
30-46% of the transferred eggs still had not expanded. This retardation is
attributed to the effects of manipulation, as suggested earlier by Tarkowski
(1959) in the case of mouse eggs. It is notable that at equivalent times treatment
groups containing a higher proportion of the more advanced stages tended to
return lower rates of viability, as judged in terms of pregnancy rate and survival
to term; compare, for example, the performance of eggs subjected to ' oestrous'
versus 'early' or 'mid-luteal' temporary recipients for 48 h (Tables 2, 3), or
108 h 'native' with 108 h 'once transferred' eggs (Tables 1, 4). One possibility is
C. E. ADAMS
Fig. 1.
Development and re-transfer of rabbit eggs
141
75 r
25 -
48
96
72
Age of egg (h p.c. plus time spent in temporary recipient)
Fig. 2. Thirty-hour eggs transferred to oestrous (O—O), early luteal (A—A) or
mid-luteal ( • — • ) temporary recipients. Sixty-hour eggs transferred to oestrous
( # — # ) or mid-luteal ( • — • ) temporary recipients. Native eggs in ligated oviducts
(x-x).
that the less well developed eggs fortuitously enjoyed a more favourable uterine
environment. However, this is unlikely because synchronization of the recipient
was arranged so as to obviate or minimize any such effect. Rather it is postulated
that the developing rabbit egg, having reached the early blastocyst stage, if
deprived of some essential factor (whether blastokinin remains an open question)
for even a short period will suffer irreparable harm. Previous observations from
this laboratory indicate that the differentiating embryonic disc is most sensitive.
When 'deprived' eggs are restored to a favourable environment (e.g. early luteal
uterus) expansion of the zona pellucida and trophoblast proliferation may still
occur though disc development fails, leading to the formation of trophoblastic
vesicles (Adams, 19706). An initial failure of the zona pellucida to expand may
FIGURE 1
(A) Early blastocyst recovered from the uterus of an untreated rabbit 84 hp.c. x 188.
(B) Early blastocyst recovered from the uterus of an untreated rabbit 96 hp.c. x 169.
(C) Early blastocyst recovered from the ligated oviduct 72 h after transfer of 30 h
eggs to a ' 1 day', synchronous temporary recipient, x 108.
(D) Early blastocyst recovered from the ligated oviduct 96 h p.c. x 143.
(E) Early blastocyst recovered from the ligated oviduct 48 h after transfer of 60 h
morulae to an oestrous temporary recipient, x 124.
(F) Herniated early blastocyst recovered from the ligated oviduct 108 hp.c. x 143.
(G) Degenerated early blastocyst recovered from the ligated oviduct 48 h after
transfer of 60 h morulae to an ' 11-day' pseudopregnant temporary recipient, x 102.
(H) Degenerate early blastocyst recovered from the ligated oviduct 120 hp.c. x 153.
142
C. E. ADAMS
be a primary factor in abnormal disc differentiation, though equally such failure
may be symptomatic rather than causal.
It appears that the rabbit blastocyst requires to undergo practically continuous
development in order to differentiate properly, any discontinuity being poorly
tolerated.
The finding that 2- and 4-cell eggs that had spent 48 h in a temporary recipient
proved more viable than those that had spent only 24 h under the same conditions
(see Table 3) reflects the greater degree of compatibility of more advanced
morulae with the uterine environment. Though the total age of the 24 h group
at re-transfer was about 54 h, the 'retarding' effect of two manipulations would
render them even less compatible. In 'straight' transfers of 2-day eggs to either
2- or 3-day uteri only 20% developed to term (Chang, 1950), whereas 2|-day
eggs were much more successful (Adams, 1962). The survival of eggs from the
48 h treatment group (Table 3), which ranged from 20 % to 40 %, was significantly lower than would be expected from 'single' transfers involving eggs of
a similar age. The explanation could be that two series of manipulations actually
have a deleterious effect on the egg, as postulated by Dickmann & De Feo (1967).
Further, the present results suggest that certain cell stages may be more affected
than others by such double manipulations.
The common occurrence of tubal ectopic pregnancy in man is exceptional;
this condition is very rare in monkeys and apparently non-existent in domestic
animals (Benirschke, 1969). Whether the human blastocyst is less demanding
in its metabolic requirements or the oviduct more able to meet blastocyst demands compared with other species is not known. Progress in the culture in
vitro of human embryos (Steptoe, Edwards & Purdy, 1971) should facilitate the
investigation of these questions.
I am indebted to Mr M. L. Norris for assistance during the course of this work, especially
with the anaesthesia.
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{Manuscript received 26 June 1972, revised 4 August 1972)