/. Embryol. exp. Morph. Vol. 23, ],pp. 21-34, 1970
Printed in Great Britain
21
The development of rabbit eggs after culture
in vitro for 1-4 days
By C. E. A D A M S 1
From the A.R.C. Unit of Reproductive Physiology and Biochemistry,
University of Cambridge
Rabbit eggs recovered from the fallopian tube during the early stages of
cleavage develop readily in culture up to the late morula stage (Lewis & Gregory,
1929; Pincus, 1930; Adams, 1956; Purshottam & Pincus, 1961; Onuma,
Maurer & Foote, 1968). The transition from morula to blastocyst, which
normally occurs 70-76 h post coitum (p.c.) coincident with entry into the uterus,
may also take place in vitro, especially if culture begins at the late morula stage.
However, growth of the blastocyst is generally limited, owing to failure of expansion of the zona pellucida which may rupture (Lewis & Gregory, 1929;
Pincus & Werthessen, 1938). Under certain conditions, prolonged blastocyst
growth even to the preimplantation stage 'with typical expansion and little
or no herniation' has been observed, though very few eggs were involved
(Pincus & Werthessen, 1938).
So far comparatively few attempts have been made to determine either the
viability or the developmental capacity of eggs after culture. Among existing
reports are those of Chang (1948), Adams (1956), Howarth, Ulberg & Alliston
(1963) and Onuma et al (1968) on the rabbit, and of McLaren & Biggers (1958),
Biggers, Moore & Whittingham (1965), Gates (1965) and Whitten & Biggers
(1968) on the mouse. The present work was undertaken in order to evaluate the
developmental capacity of rabbit morulae following 1-4 days culture. It has
been briefly referred to on a previous occasion (Adams, 1965).
MATERIALS AND
METHODS
A total of 99 young sexually mature does from our own colony was used,
25 as donors and 74 as recipients. Superovulation was induced in the donors
by treatment with a horse anterior pituitary preparation followed by mating
with fertile males, according to Adams (1962). Sixty hours p.c. the eggs were
flushed with physiological saline from the fallopian tubes (22 does) and/or the
1
Author's address: Animal Research Station, 307 Huntingdon Road, Cambridge, CB3 OJQ.
2-2
22
C. E. ADAMS
FIGURE 1
(A) Morula recovered 60 h post coitum. x 300.
(B) Early blastocyst recovered 72 h post coitum. x 330.
(C) Expanding early blastocyst recovered 84 h post coitum. x 275.
(D) Herniated early blastocyst developed from 60 h morula cultured in vitro for
48 h. x275.
(E) Herniated early blastocyst developed from 60 h morula cultured in vitro for
72 h. x275.
(F) Herniated, expanding, early blastocyst developed from 60 h morula cultured
in vitro for 96 h. x 275.
Development
of rabbit eggs after culture
23
uterine horns (3 does). The mean number of eggs recovered from the 25 donors
was 39-8 ± 2-3 (15-59). Of the total of 996 eggs recovered 949 (95-3 %) had
reached an advanced morula stage (Fig. 1A), four showed retarded cleavage
and the remaining 43 were unfertilized. Within 30-60 min of recovery more than
800 of the morulae were placed in sterile 2 ml glass tubes, at the rate of 5 per
tube. The tubes were almost completely filled with physiological saline/homologous plasma (1:1) and then tightly stoppered with silicone rubber bungs before
Utero-tubal junction
Uterus
Forceps
Blastocyst
Endometrium
Cervix
Fig. 2
being placed in an incubator at 37 °C. The plasma had been stored at - 2 0 ° C
after Seitz filtration. In one series containing 350 eggs, 1000 units penicillin
(crystalline sodium salt, Glaxo) dissolved in physiological saline were added to
each tube. At 24 h intervals, groups of eggs were removed at random from the
incubator for examination under a stereomicroscope (x 25, x 100). Depending
upon their stage of development, they were quickly classified as morulae,
unexpanded early blastocysts or expanding early blastocysts, using a normal
series as reference (Fig. 1A-C) (Adams, 1958). Careful note was made of abnormalities: for example, rupture of the zona pellucida.
Upon completion of this examination, approximately 75 % of the eggs were
transferred to the uteri of recipients which had been mated with vasectomized
males 63-97 h previously. Synchronization of the recipient's luteal stage was
arranged to coincide with the developmental stage of the eggs rather than with
the eggs' absolute age, i.e. age at recovery + time spent in culture. A description
of the egg transfer technique has been given elsewhere (Adams, 1962).
24
C. E. A D A M S
Forty-two of the recipients (group l) were allowed to survive to term so that
the numbers and condition of young, if any, could be recorded. Pregnancy was
diagnosed by palpation 7-8 days after transfer (Suitor, 1946). The remaining 32
does were killed 72 h (group 2), 96 h or 120 h (group 3) after egg transfer in order
to determine the condition of the embryos at about the time of implantation.
As quite large blastocysts and morulae were liable to be present in the same
uterine horn the following two-stage recovery technique was adopted. The
first stage, for the recovery of blastocysts, was described by Lutwak-Mann,
Boursnell & Bennett (1960); it is illustrated in Fig. 2. Medium to large-sized
blastocysts are exposed to view as the dissection proceeds but location of
smaller specimens (< 10 mm), which may lie obscured in the endometrial
folds, necessitates careful search. All blastocysts recovered at this time were
transferred to methanol. Later, they were measured and prepared for examination as flat mounts (Adams, Hay & Lutwak-Mann, 1961).
Next the uterine horn was held in the form of a semi-circle with the endometrium facing downwards towards a watch-glass (6 cm diameter), containing
6 ml physiological saline, through which it was passed about 20 times (Fig. 3).
Only the endometrium was allowed to become immersed. After standing a few
minutes any eggs present, together with debris, settle to the bottom of the watchglass. In the group 3 does some of the implantation sites were examined
histologically.
If a blastocyst could be removed intact from the endometrium, it was regarded as 'not implanted'. Conversely, if a blastocyst could not be removed
without rupture it was regarded as implanting. Usually, implanting blastocysts
had caused some distension of the uterus.
RESULTS
(1) Stage of development attained by 60 h morulae after culture in vitro
for 24-96 h
Generally no change was detected in the appearance of the culture medium;
however, in a small number of cases it became cloudy after 3-4 days incubation
Development
of rabbit eggs after culture
25
and bacteriological examination revealed the presence of the organism B.
subtilis. In the presence of this organism it was regularly found that egg development had ceased at the late morula stage, in which the blastomeres appeared
darker than normal. As none of the transfers involving these eggs proved
successful owing to infection of the uterus, they have been excluded from the
following analysis of development.
Table 1. Classification according to stage of development of 815 rabbit eggs
following culture in vitro at 37°C for 1-4 days. (No. of herniated blastocysts
in parentheses)
Early blastocysts
Culture
period
(h)
No. of
eggs
examined
Morulae
Unexpanded
No.
/o
24
48
72
96
48*
72*
96*
125
110
186
59
47
123
165
98
29
28
9
9
12
11
78-4
26-4
150
15-2
191
9-7
6-7
Expandi ng
t
No.
25
12
29
9
4
10
23(1)
/o
200
10-9
15-6
15-2
8-5
8-1
13-9
No.
2
69
129
41
34
101
131
(9)
(71)
(33)
(10)
(40)
(99)
/o
1-6
62-7
69-3
69-5
720
82-1
79-4
* With penicillin.
In Table 1, a total of 815 eggs, including 335 cultured with penicillin, is classified into the three developmental stages described earlier. After 24 h culture
most of the eggs were in the late morula stage and though some (20-0 %) had
reached the early blastocyst stage, in only two cases (1-6 %) had any expansion
occurred. During the next 24 h the position changed significantly with approximately three-quarters undergoing slight expansion, reaching the size normally
observed at about 84 h p.c. in vivo. There was practically no change between 72
and 96 h, indicating the maximal growth had already occurred during the first
48 h.
Though none of the blastocysts exceeded 200 JLI in diameter, cellular proliferation appeared to continue during the first 72 h and possibly throughout the
culture period. Herniation of the zona pellucida occurred with increasing
frequency from 48 h onwards (Fig. 1D-E). At the end of 96 h culture, approximately 80 % of the expanded blastocysts had herniated, usually at one point
but sometimes in two; occasionally a cavity was present within the herniated
mass. In the unexpanded group hernia was only observed after 96 h culture, and
it tended to be less pronounced. In one tube whose contents were examined
after 96 h incubation, two 'exblastocysts' which had arisen spontaneously and
which resembled those described by Daniel (1963) were observed. Each 'exblastocyst ' possessed a distinct cavity but there was no trace of a zona. It seems
26
C. E. ADAMS
likely that they arose through the separation of a herniated mass from the
parent blastocyst.
Development was enhanced by the inclusion of penicillin in the culture
medium both in so far as fewer morulae (7 % v. 15 %) failed to develop into
early blastocysts and more of the blastocysts subsequently underwent expansion
(80 % v. 69 %).
(2) Recipients
In the group 1, 2 and 3 recipients the mean numbers of ovulations were 11-7
(6-16), 12-8 (9-17) and 12-7 (9-16), whilst the mean numbers of eggs transferred
were 8-3 (5-12), 8-4 (6-13) and 8-9 (7-10) respectively.
(3) Transfer of eggs cultured 24-96 h
(a) In recipients allowed to survive to term (group 1). Three hundred and fifty
eggs, cultured for 24, 48, 72 or 96 h were transferred to the uteri of 42 recipients
which were allowed to survive to term. Details of the four groups including the
Table 2. Viability of eggs cultured in vitro at 37 °C for 1-4 days as
determined by survival to term following transfer to recipients
(M = morula; U = unexpanded early blastocyst; E = expanded early blastocyst)
No.
Culture
i
period
Eggs
(h)
Recipients
transferred
24
48
72
96
9
10
14
9
87(63 M ;24U)
85(13M;7U;65E)
114(3M;8U;103E)
64(2M;4U;58E)
No,
Eggs
Eggs
(
Eggs
developing
developing
Recipients
^
Young
to term producing transto term
born
young ferred
(%)
(%)
53
28
8
0
60-9
32-9
7-0
0
8
7
5
0
71
58
39
0
74-6
48-3
20-5
0
stage of egg development at transfer are given in Table 2, which shows the
proportion of eggs developing to term, first on the basis of all recipients and
second only in those recipients which maintained pregnancy to term. This latter
group decreased in proportion as the length of incubation of the eggs increased.
No control group involving the immediate transfer of eggs before culture was
included because comparable data already existed (Adams, 1962).
Considering all recipients, the proportion of eggs developing to term was
61 % after 24 h culture, comparing favourably with results obtained with noncultured eggs: under similar experimental conditions, when freshly recovered
eggs were transferred either at the rate of 5 or 10 per recipient, the proportions
surviving to term were 64 % and 46 % respectively, or 75 % and 57 %, if recipients
with implantation failure are excluded (Adams, 1962). After 48 h culture, 33 %
of eggs survived to term, decreasing to 70 % after 72 h. No young were obtained
from the '96 h group' and no recipient of such eggs was diagnosed pregnant by
Development
of rabbit eggs after culture
27
palpation 7-8 days after transfer. Excluding recipients in which pregnancy
failed, there was a steady decline in the proportion of eggs surviving to term.
In the 24 h group only morulae and unexpanded early blastocysts were transferred, but from 48 h onwards expanded early blastocysts predominated.
Though several herniated early blastocysts were transferred it is not possible
to estimate what proportion survived to term, as they were used in conjunction
with intact ones. However, in two cases it is definitely known that herniated
specimens gave rise to normal young.
Table 3. Condition of'eggs' after culture in vitro at 37 °C for 72 h or 96h
followed by transfer to recipients for 3 days
(Six recipients per group. M = unexpanded early blastocyst; E = expanded early
blastocyst. No. of herniated early blastocysts in parentheses)
Condition of blastocysts
recovered
Period of
culture (h)
72
96
96*
Condition of'eggs'
transferred
3U;42E(23)
3M;14U(9);24E(22)
18U;34E(32)
No. 'eggs'
Expanded
A
A
,
* ,
* UnexTransferred Recovered Spherical Degenerate panded
45
41
52
40
21
44
251
9%
30
15
8
5
0
4
9
Native
eggs
8
16
7
* Penicillin added to culture medium, f Four possessed an embryonic disc. X One possessed an
embryonic disc.
The interval from egg transfer to parturition varied from 29-31 days (24 h
culture), 28-34 days (48 h) to 30-34 days (72 h). To these figures should be added
2\ days, the age of the eggs at recovery, plus approximately \-\ day to cover
development in culture, in order to obtain an estimate of the length of gestation.
Whenever gestation was prolonged it was associated with a reduced litter size,
1-4 young, and stillbirths, of which there were 20 (22-5 %). Though the proportion of stillbirths is much higher than normal (Adams, 1960), it is not inconsistent with observations on small litters. No abnormal young were observed and
birth, weights were within the normal range.
(b) In recipients autopsied 3 days after transfer. One hundred and thirty-three
eggs cultured for 3 or 4 days were transferred to a total of 18 recipients which
were autopsied 3 days later. Full details are given in Table 3. Implantation had
not occurred in any recipient.
Following the transfer of eggs cultured for 3 days, 40 blastocysts (89 %) were
recovered, including 15 ' degenerate' forms (Fig. 4 A-C). Eggs cultured for 4 days
before transfer subsequently yielded 58 % of the eggs transferred: 17 blastocysts,
of which eight appeared degenerate, and four morulae. In contrast, the eggs
cultured for 4 days with penicillin gave both a higher rate of recovery, 85 %, and
28
C. E. ADAMS
a higher yield of blastocysts, 68 %. Fifty to sixty % of the eggs transferred were
herniated early blastocysts. A total of 31 native eggs, equivalent to 13-4 % of the
ovulations, was recovered.
The mean diameters of 50 of the 64 blastocysts recovered are given in Table 4.
Those developing from eggs cultured for 72 h ranged in size from 1-4 mm,
comparing favourably with blastocysts recovered from untreated does 144 h
p.c. Extending the culture period a further 24 h before transfer resulted in
slightly smaller blastocysts. Blastocyst size was not significantly affected by the
addition of penicillin to the culture medium.
Upon detailed examination the majority of the blastocysts originally thought
to be normal proved to be trophoblastic vesicles. Only five of the 64 blastocysts
possessed a disc, whose developmental stage was not readily classifiable
(Fig. 4D, E). None showed any signs of primitive streak formation, normally
present 7 days p.c. (Fig. 4G). In all of the blastocysts the trophoblast appeared
normal and showed plenty of mitotic activity. However, the extra-embryonic
endoderm was reduced in amount or absent.
(c) In recipients examined 4 or 5 days after transfer. The interval between
transfer and examination was extended by 1 or 2 days in order to provide more
time for implantation to occur and to determine whether the trophoblastic
vesicles could implant. The results are summarized in Table 5. Implantation was
taking place in six of the recipients, in which large unattached blastocysts were
also found. In the does examined 4 days after receiving morulae that had been
cultured for 2, 3 or 4 days, three out of four, two out of six and zero out of 15
respectively of the blastocysts possessed a disc which varied from 340 x 360 [i to
1015 x 1270 ju, in size (Fig. 4F); blastocyst development corresponded with the
normal 6-6^ day stage, indicating retardation since the total age of the embryos,
including the culture period, was 8^-9| days. In each of the three implantation
sites examined histologically the embryo appeared normal (Fig. 4H, I). None
of the trophoblastic vesicles had implanted. The decline both in the proportion of
implantations and of 'embryos' recovered between the 4th and 5th days suggests that the most critical stage occurs about this time.
FIGURE 4
(A-C) Degenerate blastocysts recovered from uteri of group 2 recipients 72 h after
transfer of eggs cultured for 72 h (A) or 96 h (B and C). x 50.
(D-E) The embryonic disc of blastocysts recovered from the uteri of a group 2 recipient examined 72 h after transfer of eggs cultured for 72 h. D, x 150; E, x 60.
(F) The embryonic disc of a blastocyst recovered from a group 3 recipient examined
4 days after transfer of eggs cultured for 48 h. x 160.
(G) The embryonic disc of a blastocyst recovered from an untreated rabbit 7 days
p.c. x 60.
(H)T.S.of an implantation site in a group 3 recipient autopsied 96 h after the transfer
of eggs cultured for 48 h. x 10.
(1) Higher power view of the same implantation site to show the attachment of the
trophoblast to the endometrium, x 130.
Development of rabbit eggs after culture
, -; w # u
H
/
29
30
C. E. ADAMS
Table 4. Diameter of blastocysts resulting from morulae cultured in vitro for
72 or 96 h before transfer to the uteri of pseudopregnant recipients for 72 h
{means ±S.E., range in parentheses)
Culture
period
(h)
No. of
blastocysts
72
96
96*
24
9
17
Diameter (mm)
Long axis
2-87±0-16 (1-04-3-95)
2-00±0-21 (1-41-2-71)
2-13 ±013 (1-30-3-07)
Short axis
2-73 ±0-16 (0-94-3-78)
1-78 ±0-22 (0-99-2-57)
1-97 ±0-13 (1-27-2-93)
* Penicillin added to culture medium.
Table 5. Transfer of eggs after culture in vitro for 48, 72 or 96 h to the uteri of
pseudopregnant rabbits: autopsy 96 or 120 h after transfer
(Herniated early blastocysts in parentheses)
Condition of embryos at recovery
Autopsy
(h after
Culture
egg transfer) period (h) Recipients
48
96
72
96
72
120
96
No.
eggs
transferred
19(1)
29 (15)
27 (24)
35(11)
12(5)
Blastocysts
Implanting Un implanted Degenerate
1*
5
10
4*
12
1
2*
4
12 1*
0
0
7
Burst when uterus was opened.
DISCUSSION
In the present experiments a high proportion of morulae, cultured in vitro for
1-4 days at 37 °C, developed into early blastocysts, confirming the earlier
findings of Lewis & Gregory (1929), Pincus & Werthessen (1938) and Purshottam & Pincus (1961). However, these early blastocysts failed to show the marked
expansion recorded by Pincus & Werthessen in two of their three culture
systems. These systems differed from the present principally in the greater
volume of medium used, and in one series the possession of means for its circulation. Normally the rabbit blastocyst grows rapidly from 80 h onwards and the
zona stretches to accommodate it. This the zona failed to do under present
conditions. When Lewis & Gregory (1929) observed a similar failure they
postulated that 'it is probable that some of the secretions of the uterus alter the
characteristics of the zona pellucida'. In this connexion it is especially significant
that eggs which failed to expand fully in vitro nevertheless resumed growth and the
zona became attenuated following transfer to the uterus of pseudopregnant
recipients. This proves that the zona had not lost its capacity to expand but
Development of rabbit eggs after culture
31
rather that conditions in vitro were not conducive to expansion. Recently,
evidence has been presented that a uterine protein fraction, termed 'blastokinin', which may be identical with 'uteroglobin' described by Beier (1968), can
induce and regulate blastulation (Krishnan & Daniel, 1967). However, since
Pincus & Werthessen (1938) found that the zona could expand under certain
conditions in vitro this implies that specific uterine factors are not absolutely
essential, at least for the expansion of the blastocyst. Nevertheless, the fact
that expansion in vitro is slower than in vivo and that development may not be
normal does attest to the significance of the uterine environment. The observation
that 60 h morulae do not expand fully when transferred to the uterus on the eighth
day of pseudopregnancy (Adams, 1968) provides evidence that uterine conditions favouring expansion (presence of blastokinin ?) are short lived.
Lewis & Gregory (1929), with the aid of cinematography, observed that
herniation of the zona might occur repeatedly in the same blastocyst, the cells
of the trophoblast apparently healing over the rupture points. This process
must also have occurred following the transfer of several herniated early blastocysts (Table 3) since 3 days afterwards no sign of herniation was present in the
now expanded blastocysts. Moreover, at least two and possibly several more
herniated blastocysts developed into term foetuses.
Transfer of cultured eggs to pseudopregnant recipients which were allowed
to survive to term showed that over the 4-day culture period the eggs' developmental capacity fell steadily from the control level of approximately 60 %
(Adams, 1962) to zero. Furthermore, the proportion of recipients in which
implantation failed, as determined by palpation, also increased over the same
period. This indicated either that some of the embryos were already non-viable
at the time of transfer or that they possessed only a limited developmental
capacity. That the second explanation was, in fact, more valid became apparent
when examinations were made within a few days of transfer, about the time of implantation. Although a considerable proportion of apparently normal blastocysts
was recovered at this time more detailed examination revealed that few possessed
an embryonic disc and most were, in fact, trophoblastic vesicles. Previously,
similar vesicles have been described developing from single blastomeres of the
2- or 4-cell stage of rabbit eggs (Seidel, 1956; 1960) and mouse eggs (Tarkowski,
1959 a, b; Tarkowski & Wroblewska, 1967), as well as following the re-transfer
of rat blastocysts which had spent 3-4 days in the oviduct of mice (Tarkowski,
1962). It is notable that such hollow rat blastocysts did not differ from normal
ones in their ability to elicit a decidual reaction. In the present series, however,
no evidence was obtained to suggest that the vesicles without discs could
implant.
SUMMARY
1. Fertilized rabbit eggs recovered 60 h p.c. from the fallopian tubes or uteri
of superovulated rabbits were cultured at 37 °C in homologous plasma/physio-
32
C. E. A D A M S
logical saline (1:1) with or without penicillin for periods of up to 96 h. The
eggs' development was recorded after 24, 48, 72 or 96 h in culture.
2. After 24 h one-fifth of the morulae had developed into unexpanded early
blastocysts, and by 48 h approximately two-thirds had undergone expansion,
the largest reaching 200/* in diameter and this diameter was not exceeded in
the 72 and 96 h groups. Rupture of the zona pellucida occurred increasingly
from 48 h onwards so that at 96 h 75 % of the blastocysts had ruptured zonae.
3. A total of 350 eggs cultured for 24, 48, 72 or 96 h was transferred to the
uteri of 42 pseudopregnant recipients which were kept to term. The yield of
young fell from 61 % with eggs cultured for 24 h to zero with eggs cultured for
96 h. The proportion of recipients pregnant when examined at 10 days similarly
decreased, from 89 % to zero. No gross abnormalities were observed in the
89 young but 20 were stillborn. Stillbirths were associated with small litters
and extended gestations.
4. In eighteen recipients examined 3 days after the transfer of 138 eggs cultured for 72 or 96 h implantation had not occurred. Of the 92 blastocysts
recovered, 28 showed marked degeneration and only five of the remaining 64
possessed a disc. The 59 trophoblastic vesicles resembled normal 6-day blastocysts in size.
5. In a further 14 recipients examined 4 or 5 days after transfer, a few implantation sites were found: those examined histologically contained embryos.
There was no evidence that trophoblastic vesicles could implant. The proportion
of blastocysts to trophoblastic vesicles recovered decreased as the period of
culture increased.
RÉSUMÉ
Le développement d'œufs de lapin cultivés in vitro pendant 1 à 4 jours.
1. Des œufs tubaires ou utérins de lapin âgés de 60 h sont cultivés à 37 ° C dans
du plasma homologue et une solution saline (1/1) avec ou sans pénicilline pendant 24, 48, 72 ou 96 h.
2. Au bout de 24 h, 1/5 des morulas deviennent de jeunes blastocystes. Au
bout de 48 h, 2/3 environ deviennent des blastocystes dilatés jusqu'à 200 pi de
diamètre. A partir de 48 h la membrane pellucide se rompt, jusqu'à 75 % des cas
au bout de 96 h.
3. Trois cent cinquante œufs ont été cultivés pendant 24, 48, 72 ou 96 h et
transférés dans l'utérus de 42 femelles pseudogestantes. Le rendement des
naissances tombe de 61 % pour des œufs cultivés 24 h à 0 % pour des œufs
cultivés 96 h. Au bout de 10 jours le pourcentage des femelles gravides décroit
de 89 % à 0 %. Sur 89 jeunes on observe peu de grosses anomalies mais 20 mortsnés, associés avec des portées réduites et un allongement de la gestation.
4. Chez 18 femelles examinées au bout de 3 jours après avoir reçu 138 œufs
cultivés 72 à 96 h, il n'y a pas d'implantation. Sur les 92 blastocystes recueillis,
Development
of rabbit eggs after culture
33
28 ont dégénéré et cinq seulement possèdent un bouton embryonnaire. Pour
59 vésicules trophoblastiques l'aspect est celui de blastocystes de 6 jours.
5. Chez 14 femelles examinées 4 à 5 jours après le transfert des œufs, on
observe quelques implantations contenant des embryons. Il n'est pas évident
que les vésicules trophoblastiques puissent s'implanter. Le nombre des vésicules
trophoblastiques décroit en raison inverse de la durée de la culture.
I am indebted to Dr Mary F. Hay for help in preparing and interpreting the blastocyst flat
mounts, and to Mr M. L. Norris for technical assistance.
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