Reprod Dom Anim 45, 760–772 (2010); doi: 10.1111/j.1439-0531.2009.01345.
x
ISSN 0936-6768
Morpho-physical Recording of Bovine Conceptus (Bos indicus) and Placenta from
Days 20 to 70 of Pregnancy
AC Assis Neto1, FTV Pereira1, TC Santos2, CE Ambrosio3, R Leiser4 and MA Miglino3
1
Campus Experimental de Dracena, São Paulo State University, Dracena, São Paulo, Brazil; 2Department of Animal Science, Universidade Estadual
de Maringá, Maringá, Brazil; 3Surgery Department of School of Veterinary Medicine and Animal Science, Sao Paulo University, São Paulo, Brazil;
4
Department of Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University, Giessen, German
Contents
The study is based on 141 pregnant Bos indicus cows, from
days 20 to 70 post-insemination. First, special attention was
given to the macroscopically observable phenomena of
attachment of the conceptus to the uterus, i.e. the implanta-
tion, from about days 20 to 30 post-insemination up to day 70,
and placentome development by growth, vascularization and
increase in the number of cotyledons opposite to the endome-
trial caruncles. Secondly, as for the conceptuses, semiquanti-
tative, statistical analyses were performed of the lengths of
chorio-allantois, amnion and yolk sac; and the different parts
of the centre and two extremes of the yolk sacs were also
analysed. Thirdly, the embryos ⁄ foetuses corresponding to
their membranes were measured by their greatest length and
by weight, and described by the appearance of external
developmental phenomena during the investigated period like
neurulation, somites, branchial arcs, brain vesicles, limb buds,
C-form, pigmented eye and facial grooves. In conclusion, all
the data collected in this study from days 20 to 70 of bovine
pregnancy were compared extensively with corresponding data
of the literature. This resulted in an ‘embryo ⁄ foetal age-scale’,
which has extended the data in the literature by covering the
first 8 to 70 days of pregnancy. This age-scale of early bovine
intrauterine development provides model for studies, even
when using slaughtered cows without distinct knowledge of
insemination or fertilization time, through macroscopic tech-
niques. This distinctly facilitates research into the cow, which
is now being widely used as ‘an experimental animal’ for
testing new techniques of reproduction like in vitro fertiliza-
tion, embryo transfer and cloning.
Introduction
The bovine embryo, in recent times, after the advent of
techniques like in vitro fertilization (Farin and Farin
1995) and cloning (Hill et al. 2000; Smith and Murphy
2004), has become of more value. The bovine embryos
can be flushed out from the uterus relatively easily
from the beginning of pregnancy up to several weeks
(Newcomb et al. 1978; Maddox-Hyttel et al. 2003). The
conceptus can be observed in situ after the excision of
the uterus from the cows killed in slaughterhouses or
they can be taken out from the uterus for further
experimental use (Leiser 1975; King et al. 1979).
The morphology of bovine embryos is particularly
well known up to day 8 post-insemination (p.i.) as long
as the zona pellucida is intact (Winters et al. 1942),
allowing the embryo to be easily manipulated for
research (Maddox-Hyttel et al. 2003). During the elon-
gation phase of embryogenesis (Betteridge et al. 1980)
from day 12 to about 20 p.i., studying the morphology
of the embryo and its membranes in toto is complicated
(Maddox-Hyttel et al. 2003). During this time, the
chorionic sac may grow from a few centimetres up to
30 cm or even more (Winters et al. 1942; Chang 1952),
depending on the age and other embryo-related charac-
teristics or external influences from the mother (Winters
et al. 1942). In addition, the tissue of the conceptus,
during this period, is very delicate to handle and cannot
be isolated from the uterus because of the enormous
extension inside the uterus to which it clings but is not
fixed (Winters et al. 1942). Other critical factors are the
thinness and the high water content of this tissue (R
Leiser unpubl. obs.).
Not much is known of the morphology of the embryo
during the implantation period from day 20 to about
day 33 p.i. (Leiser 1975; King et al. 1980). Leiser (1975)
showed the sequential stages of precontact, apposition
and adhesion between the uterus and the chorion,
ending in a microvillous interdigitation of the uterine
epithelium and trophoblast in the caruncular and
intercaruncular regions as well. During this period of
implantation, the conceptus can barely be flushed out
from the uterus for experimental use (Leiser 1975).
Gestation length varies among breeds, with Bos
indicus cows tending to have longer gestation lengths
(284 days to more than 300 days) than do Bos taurus
cows (generally 280 to 285 days) (Paschal et al. 1991;
Chenoweth 1994; Wooding and Flint 1994; Cavalcante
et al. 2001); however, not much has been known
regarding the differences, if any, in the placental
development between these two bovine species. In this
study, the periods of implantation and placentome
formation 20 to 70 days p.i. in B. indicus related to the
chorion, including the developmental characteristics of
amnion, yolk sac and allantois were investigated by
photography and by measurements like length, width
and weight measurements, and the amount of caruncles.
Specifically, systematic macroscopy was performed on
embryos and foetuses inclusive of placental membranes,
which were flushed or cut out from uterus or manually
prepared separately from the endometrium after having
cut open the uteri on their dorso-antimesometrial side.
Material and Methods
Sample collection
This study was based on uteri of 141 pregnant B. indicus
cows. The samples were collected from the slaughter-
house at Dracena, Sao Paulo, Brazil. Immediately
after the slaughtered of the cows, each uterus was
removed and conceptus was separated for examination.
The experimental protocol (number 631 ⁄ 2005) was
 2009 Blackwell Verlag GmbH
Macroscopy of Bovine Embryo ⁄ Foetus and Membranes
authorized by the Bioethics Committee of the School of
Veterinary Medicine, University of Sao Paulo (USP),
Brazil.
Determination of gestational period
The early gestational period between days 20 and 70 p.i.
of pregnancy was determined according to the exterior
characteristics of the embryo ⁄ foetus (Fig. 1) – greatest
length (GL; Winters et al. 1942); as well as develop-
mental morphology of embryonal ⁄ foetal membranes
(Table 1). Based on established criteria, the embryos
and foetuses were included in six day-groups of preg-
nancy: 20 to 25 (n = 26), 25 to 30 (n = 23), 30 to 40
(n = 25), 40 to 50 (n = 24), 50 to 60 (n = 21) and 60
to 70 (n = 22).
Macroscopic and morphometric analysis
The whole macroscopical preparative process of the
material in this study was photographed for recording
the structural characteristics. The photographic camera
used was Leica (Model MDa; Ernst Leitz, Wetzlar,
Germany). Illumination was provided by photoflood-
light (250 W) from upward to downward and blue
counterlight (40 W) from downward to upward through
a glass container, which contained the specimen
immersed in water. The uteri were collected and the
uterine horns opened as well as dissected at their dorso-
antimesometrial line in order to expose the embryo-
nal ⁄ foetal membranes. The chorionic membrane or
gestational sac was carefully cut together with the
allantois, amnion, and yolk sac inside, which were
measured using a paquimeter. The variables of mea-
surement of all these membranes were the lengths
(cranio-caudal), and the width as concerning the
amnion. Of special interest, in addition, was the number
of cotyledons on the chorion. Finally, the GL of embryo
and foetus was determined and their weight estimated
using an analytical scale (0.001 to 200 g). The measure-
ments were ascertained after the specimens were fixed in
10% formaldehyde in order to solidify the tissue, and
for histological use (in a different study).
Statistical analysis
The analyses were conducted by STATISTIC-R software
(R Development Core Team 2006: A Language and
Environment for Statistical Computing). The qualitative
(a)
Fig. 1. (a) Bovine embryo viewed
from left and presented in this
study as a model to show external
characteristics. 1.2 cm GL (greatest
length), 32 days. (b) Schematic
drawn of the embryo: legends to A:
GL = dashed line, 1, forebrain; 2,
pigmented eye; 3, cranial neural
pore; 4, branchial arcs; 5, cervical
flexure; 6, somites; 7, tail; 8, bud of
foreleg; 9, bud of hindleg; 10,
heart; 11, liver. Scale bar 0.5 cm
 2009 Blackwell Verlag GmbH
761
parameter from the number of cotyledons referring to
the different gestational periods was analysed by ‘box-
plots’. Descriptive statistical analysis was performed in
order to calculate the mean and standard deviation of
the length of the foetal membranes, and the GL and
weight of embryos ⁄ foetus.
Results
The ‘reactive appearance’ of the conceptus inside the
uterus is distinctly different, depending on the stage of
pregnancy, which is evident when the uteri are opened.
Because of this phenomenon, we were forced to collect
and methodically handle the conceptuses differently
depending on the stage of pregnancy; hence the samples
are presented in three groups here.
External morphological characteristics of embryo ⁄ foetus
and corresponding membranes (Table 2)
Conceptuses of days 20 to 30 of pregnancy
Single bovine embryos were identified, faintly shining
through the embryonic membranes located in one of the
opened uterine horns. The chorion – wrinkled, trans-
parent, and ending in two thin processes–extended
throughout the whole uterine lumen at day 23 of
pregnancy (Fig. 2). But at its largest part the chorion
enclosed the embryo which, together with the amnion
surrounding it tightly, marks the embryonic zone in the
so-called ‘pregnant uterine horn’ (Fig. 2). The allantois
was separated from the chorion, but its stalk leading
ventrally out from the embryo was well visible at day
23 p.i. and later (Figs 2–4). An initial vasculature was
obvious, particularly along the mesometrially oriented
border of allantois from about day 26 p.i. (Figs 3 and 4).
The yolk sac which arises from the ventral part of the
embryo (Fig. 3) was prominent and composed of a
central part with two slender peripheral projections of
different lengths (Figs 3 and 4; Table 3), both of which
diverge into the extremes of the chorionic sac. The
embryo shows the typical C-shaped body form during
this period (Figs 4 and 6).
Conceputses of days 30 to 50 of pregnancy
During this time the conceptus appears sausage-like in
the dorsally opened uterine horn. The chorionic surface
appears smooth with no macroscopically detectable
(b)
 Table 1. Age ⁄ greatest lengths1 timetable of bovine development at days 8–70 with respect to distinct external morphological characteristics of conceptus and foetal membranes 762

Greatest Length or
Approximative Lengths of maximal
time of Embryo ⁄ elongation of Insemination
pregnancy Foetus, mm1 chorion, cm External characteristics of bovine Embryo ⁄ Foetus membranes Authors method used

8d 0.196 Hamilton and Laing 1946

8 d 14 h Hatching of embryo or disappearance of zona pellucida Winters et al. 1942 a
9 d 19 h Germ disc visible Winters et al. 1942 a
10 d 0.3 Betteridge et al. 1980 c
11 d 0.385 Chang 1952 d
0.818 First stage of chorion elongation Betteridge et al. 1980 c
12 d 2.78 Betteridge et al. 1980 c
13 d 2.8 Last unhatched embryo observed Betteridge et al. 1980 c
14 d 0.1–0.44 0.5–1.9 Amnion folding Maddox-Hyttel et al. 2003 c
5.7 Betteridge et al. 1980 c
14 d 14 h Chorion extension in 2 ⁄ 3 of pregnant uterine horn2 Winters et al. 1942 a
15 d 13.5 Betteridge et al. 1980 c
16 d 0.62 Blastocyst like ribbon Chang 1952 d
15 Enormous logarithmic elongation of chorion Betteridge et al. 1980 c
17 d 16 Primitive streak observable, beginning of neural folding to neural groove Chang 1952 c
17 d 14 h Extension of chorion throughout the whole pregnant uterine horn2 Winters et al. 1942 a
18 d Beginning of implantation (days 16–18) Greenstein et al. 1958 a
with apposition phase of materno-foetal microvillous interdigitation Leiser 1975 b
19 d Amnion complete Evans and Sack 1973
19 d 14 h 2.35 Embryo shows head fold, extension of chorion starting into ‘‘unpregnant’’ Winters et al. 1942 a
uterine horn
19 d 19 h Neural folds closed in mid-region, five somites Winters et al. 1942 a
20 d 14 h Eight pairs of somites (somites grow very fast) Winters et al. 1942 a
21 d 2.86 (0.78–5.89) 30 Neural tube complete, 14 somites, allantoic bud Maddox-Hyttel et al. 2003 c
22 d Implantation with adhesion phase ending in the embryo zone of uterine horn Leiser 1975 b
Yolk sac distinct in three parts: vitelline duct, centre, 2 projections Rüesse et al. 1992
22 d 16 h 5.18 Torsio of embryo, optic and otic vesicles, first branchial arc, heart prominence, Winters et al. 1942 a
distinct development of allantois
23 d 40 Allantois stalk distinct, chorion in both uterine horns Wrobel and Suess 1998 b
24 d Chorion in top of ‘‘unpregnant’’ uterine horn Chang 1952 d
24 d 16 h 3.713 Winters et al. 1942 a
25 d 3.5 Jost and Prepin 1966
Three brain vesicles, bud of forelimb, second branchial arc Wrobel and Suess 1998 b
Embryo C-shaped, third branchial arc Evans and Sack 1973
26 d Fourth branchial arc, hindlimb bud Evans and Sack 1973
26 d 16 h 5.58 Winters et al. 1942 a
27 d Implantation by adhesion phase finished throughout both uterine horns Leiser 1975 b
27 d 14 h 9.73 Winters et al. 1942 a
28 d 8 Jost and Prepin 1966
29 d 14 h 9.7 44.3 Melton et al. 1951
30 d 10 Jost and Prepin 1966
10.5 First very fragile cotyledons observed Melton et al. 1951
Olfactory pits forming, eyes pigmented, hand plate present Evans and Sack 1973
30 d 11 h 12 Winters et al. 1942 a
31 d 11 Jost and Prepin 1966

 2009 Blackwell Verlag GmbH

AC Assis Neto, FTV Pereira, TC Santos, CE Ambrosio, R Leiser and MA Miglino
 Table 1. (Continued)

Greatest Length or
Approximative Lengths of maximal
time of Embryo ⁄ elongation of Insemination
pregnancy Foetus, mm1 chorion, cm External characteristics of bovine Embryo ⁄ Foetus membranes Authors method used

31 d 10 h 10.3 38.4 Melton et al. 1951

32 d 9 Wrobel and Suess 1998 b
12 Jost and Prepin 1966
Beginning of placentome development (Leiser this study) by attachment of Zietzschmann 1924
chorionic villi in the endometrium (villous indentation)
32 d 12 h 12.4 45 Melton et al. 1951

 2009 Blackwell Verlag GmbH

32 d 14 h 11.9 Winters et al. 1942 a
33 d 12.5 Jost and Prepin 1966
First distinct foetal villi of cotyledon in placentome King et al. 1979
33 d 12 h 14.6 50 Fragile materno-foetal attachment on placentome Melton et al. 1951
34 d Facial clefts closed, grooves betw. forelimb digits Evans and Sack 1973
34 d 13 Wrobel and Suess 1998 b
Kritzenberger and Wrobel 2004
35 d 13.5 Wrobel and Suess 1998 b
35 d6h 15.2 61.2 17–25 cotyledons Melton et al. 1951
36 d 14 Wrobel and Suess 1998 b
Macroscopy of Bovine Embryo ⁄ Foetus and Membranes

36 d 18 h 14.7 71.3 25 cotyledons Melton et al. 1951

19 Kritzenberger and Wrobel 2004 b
37 d 3 h 18.26 Extension of body wall to the umbilicus Winters et al. 1942 a
38 d 17 Wrobel and Suess 1998 b
Eyelids forming, genital tubercle present, grooves between hindlimb digits Evans and Sack 1973
38 d 12 h 18.7 81.1 33 cotyledons Melton et al. 1951
39 d 18 ⁄ 19 Wrobel and Suess 1998 b
40 d 2 h 22.8 Winters et al. 1942 a
Sexual differentiation Wrobel and Suess 1998 b
43 d 20 Kritzenberger and Wrobel 2004 b
45 d 25 ⁄ 34 Kritzenberger and Wrobel 2004 b
4
30.8 Beginning change from embryo to foetus Winters et al. 1942 a
Tongue visible, digits separated distally Evans and Sack 1973
50 d Eyelids begin to cover eyes Evans and Sack 1973
35 Kritzenberger and Wrobel 2004 b
38.5 Elongation of neck and rotation of head 90 at the line of neck to head axis Winters et al. 1942 a
53 d 42 75 This study
55 d 46.7 Winters et al. 1942 a
60 d Eyelids fused, external genitals differentiated, hoofs forming, horn buds appear Evans and Sack 1973
66 Winters et al. 1942 a
Kritzenberger and Wrobel 2004 b
70 d 94 Distinct changes of body proportions Winters et al. 1942 a

The method of insemination used by the authors: a, natural mating under control; b, instrumental insemination; c, superovulation; d, from abattoir without control of insemination.
1
Greatest length = greatest diameter of conceptus head and rump together.
2
Length of uterine horn 30–40 cm.
3
Because of C-formation of the embryo the greatest diameter becomes reduced.
4
A slow process!
Definition: Embryo = sprouting, forming; Foetus = proportioning of body form.
763
 764

Table 2. Macroscopic morphology of bovine embryonic ⁄ foetal membranes during the days 25–65 of pregnancy

Days of pregnancy

Parameters assessed 25 days 30 days 37 days 45 days 50 days 65 days

GL (mm) 0.71 ± 0.17 1.23 ± 0.13 1.90 ± 0.27 2.79 ± 0.19 3.76 ± 0.52 6.75 ± 0.66
Chorion and Absent Absent Present only at Present throughout Present throughout Present throughout
allantois areas around the membranes and easily membranes not membranes not separable
membranes embryos separable easily separable
fusion
Allantois and Absent Absent Present only at the Present Present Present and prominent
amnion lateral part of the
membranes amnion
fusion
Chorion and Absent Absent Absent Absent Absent Starting gelatinous connection
amnion
membranes
fusion
Development Absent Absent1 Discrete in embryo Present around the Present around the Present throughout the GS,
of cotyledons region embryo and absent embryo and discrete villous substructure visible
on the GS extremities on GS extremities
Allantois Discretely Near embryo Not visible in the GS Present, but more Present and prominent Present and prominent
vascularization present mesometrially areas of peripheral distinct mesometrially (not in chorionic tips)
mesometrially distinct and thirds (of uterine horns)
near embryo antimesometrially
discrete
Chorion Absent Absent Absent and discrete Absent and discrete Present Present and prominent
vascularization
Amnion2 Absent Absent Absent Present and discrete Present Present and prominent
vascularization
Yolk sac Prominent in Distinctly developed No growth in relation to No growth (involution) Macroscopically barely Macroscopically not detectable
development all parts: duct, in relation to embryo embryo to see (involution)
centrum, 2 projections,
in total 4.5 · length
of embryo

GL, greatest length; GS, gestational sac: it fully occupies both uterine horns in the period investigated.
1
Histologically beginning of placentome development.
2
Amnion: distinct throughout the whole period investigated. Tight to embryo up to day 40, then distinct growth.

 2009 Blackwell Verlag GmbH

AC Assis Neto, FTV Pereira, TC Santos, CE Ambrosio, R Leiser and MA Miglino
Macroscopy of Bovine Embryo ⁄ Foetus and Membranes 765

E
AL AL
Fig. 2. Embryo 0.56 cm CR, E
23 days. The chorionic sac (C), AM
probably extending in full length AL
inside the uterus (44 cm), slightly is
constricted at half of its length
(arrow) which corresponds to the
C
area of corpus uteri. The embryo is
located on the largest part of
chorion which marks the area of
‘pregnant uterine horn’. Inside the E
chorion, the embryo (E) is sur-
rounded by the amnion (AM) and
linked to the allantois (AL) as
specifically shown with the small-
illustrated preparation and the
drawing. Note the two chorion tips
corresponding by location to the
extremes of uterine horns. Scale
bar 2 cm

Table 3. Length (cm) of yolk sac in bovine placenta with central part,
longer and shorter projections, from 20 to 70 days, characterizing
specifically also the involution of this membrane

C Yolk sac length (cm)

Gestational
period Longer Shorter
(days) Central projections projections Total

20–25 0.76 ± 0.24 5.14 ± 2.88 3.17 ± 1.60 9.08 ± 4.45

25–30 0.67 ± 0.16 3.90 ± 1.50 2.20 ± 1.10 6.76 ± 2.15
30–40 0.71 ± 0.18 3.01 ± 1.80 1.90 ± 1.40 5.53 ± 3.14
YP YC 40–50 0.60 ± 0.15 0.60 ± 0.80 0.33 ± 0.45 1.53 ± 1.33
50–60 0.46 ± 0.10 – – 0.46 ± 0.10
60–70 0.30 ± 0.09 – – 0.30 ± 0.09
Fig. 3. Embryo 0.6 cm CR, 26 days-surrounded by the faint amnion
(arrow), shows the yolk sac with a central part (YC) from which two
thin peripheral projections (YP, only one visible) diverge into the
chorion (C, here back reflected to the embryo). The allantois clearly
develops first blood vessels particularly along its mesometrial border
(arrowheads). Scale bar 1 cm
C C

YC

YP YP Fig. 5. Embryo 1.03 cm CR, 30 days. Allantoic blood vessels, shining

Fig. 4. Embryo 0.76 cm CR, 27 days (with the chorion and the
amnion removed). The yolk sac is distinct with a central (YC) and two
projecting (YP) parts. Two allantoic blood vessels, arising from the
embryo caudally of the yolk sac (arrow), mark the mesometrially
oriented border of allantoic sac (arrow heads). Scale bar 1 cm
connection to the uterine caruncles before about day 37
of pregnancy (Figs 5 and 7). Later, however, cotyledons
get more and more visible by increasing in number, size
and thickness (length of chorionic villosity); they
become more reddish in colour because of the increased
through the sausage-like chorion (C), by bundles extend along the
mesometrial side of conceptus (arrows). They contain branchings
which ramify by right angles (arrow heads) running to the
antimesometrial or dorsal side of embryo. The chorion does not show
any cotyledons. Scale bar 1 cm
allantoic vascularization (Figs 7–9). Cotyledonary for-
mation spreads out progressively from the chorionic
surface of the embryonic zone to the chorionic tips, but
does not build up a tight chorio-endometrial connection
until day 50 p.i. at least (Table 2). Visible through the
transparent chorion, the amnion, separated by its fluid-
filled cavity from the embryo, only slowly increases in

 2009 Blackwell Verlag GmbH

766 AC Assis Neto, FTV Pereira, TC Santos, CE Ambrosio, R Leiser and MA Miglino

run into the allantamnion, being macroscopically visible

YP
YC
Fig. 6. Embryo 1.25 cm CR, 33 days is uncovered from the amnion,
allantois and chorion (below). The yolk sac in full extension is visible
with central part (YC) and two projections (YP). Allantoic vessels,
particularly bundled in mesometrial location of the embryo (brackets),
originate by umbilical arteries (the left one clearly visible). Note the
beginning pigmentation of eye (arrow). Scale bar 1 cm
size in relation to the embryo during the beginning of
this period (Figs 5 and 7), but growth is more distinct
from day 40 of pregnancy (Figs 8, 9 and 11). The
allantois is also visible through the chorion thanks to its
blood vessels, which begin to progressively branch in the
mesometrial direction from the mesometrially located
vascular bundle to form the chorioallantoic vessel
system (Figs 5–9). From this system the vessels also
for the first time on day 45 p.i. (Fig. 11). Allantoic blood
vessels help to understand when the outer side of the
allantois fuses to the inner side of the chorion: on day 30
p.i., even though fusion is evident by histology (see
section ‘Discussion’) macroscopy is not adequate to
show fusion when, after opening of the chorion, these
vessels can be observed to be distinctly belonging to the
allantois (Fig. 7). However, from about day 37 p.i.,
these vessels can be found to be a part of the inner side
of chorion after careful manual separation of the weakly
fused chorion and allantois (Fig. 7). From day 40 p.i.
onwards, this fusion becomes progressively solid from
the embryo zone peripheral to the tips of the gestational
sac. When the eyes of the embryo begin to acquire
pigmentation at about day 30 p.i., the bundle compris-
ing the allantois, the yolk sac and the ventral amnion
wrapping them, becomes longer, thereby forming the
umbilical cord (Figs 6, 8–11), which also can been seen
macroscopically (Figs 8 and 9). The yolk sac is most
evident with its central part and two projections on day
33 p.i. (Fig. 6); it becomes smaller progressively after-
wards (Figs 10 and 12).
Conceptuses of days 50 to 70 of pregnancy
Around day 50 p.i. the conceptus changes slowly from
embryo to foetus (compare Figs 10 and 12), the latter
being typical with an elongated neck and the axis of the

Fig. 7. Embryo 1.88 cm CR,

37 days. The chorion easily can be
manually separated (C) from the
allantois (dashed janus-arrow).
However, by this, the allantoic
blood vessels ‘stick’ to the chorion
inner (mesenchymal) side (arrows).
Cotyledons (foetal part of placen-
tomes) in stage of beginning
development on the outside of
chorion can be seen shining
through the allantois (dashed
circles). The amnion is small here
(AM), therefore located very
tightly to the embryo. Scale bar
1 cm

Fig. 8. Embryo 2.17 cm CR,

41 days. Central part of conceptus
with embryo and amnion sac
(AM), viewed from the stretched
chorionic outer surface which
shows some cotyledons (dashed
circles). Note the allantoic vascu-
lature, with origin from the umbi-
licus (*), is bundled along the
mesometrial or ventral side of
conceptus giving off a rather dense
network of tributary vessels to the
dorsal side of allantochorion
(dashed arrow). Scale bar 1 cm

 2009 Blackwell Verlag GmbH

Macroscopy of Bovine Embryo ⁄ Foetus and Membranes
Fig. 9. Embryo 2.77 cm CR,
45 days. In comparison to Fig. 8
the amnion is larger, and the
cotyledons are better visible
(dashed circles) because of
increased vasculature and irriga-
tion by blood. Scale bar 1 cm
Fig. 10. Embryo out of Fig. 9. The chorioallantois and the amnion
have been manually deplaced to the bottom of figure. The relatively
small yolk sac with its central part (arrow) arises from the cranial part
of umbilical cord. Scale bar 1 cm
Fig. 11. Embryo 2.72 cm CR, 45 days being by age comparable to the
embryos of Figs 9 and 10. Note the distinct allantoic vasculature with
origin out of the umbilicus running along the mesometrial border
of chorioallantois (below). It gives rise to the first blood vessels
macroscopically visible in the (allant-) amnion (arrow). Scale bar 1 cm
neck bent by about 90 to the axis of the head. The
formation of a long foetal splanchnocrane is conspicu-
ous in bovine species.
The chorioallantois is fixed inside the opened uterus,
but can be manually separated between the cotyledons
and caruncles of placentomes, quite easily around day
50, which can be more difficult at around day 70 p.i..
Taken this way out from the uterus, the sausage-like
chorioallantoic sac filled with allantoic fluid shows
distinct asymmetry by length and width, i.e. a longer
wider part and a shorter narrower part, both separated
 2009 Blackwell Verlag GmbH
767
Fig. 12. Foetus 4.4 cm CR, 54 days. The umbilical cord is distinct by
allantoic duct (not visible here) and blood vessels (arrowhead: an
umbilical artery). The yolk sac in stage of involution barely is visible
(arrow). Scale bar 1 cm
by a distinct constriction which mirrors the uterine body
between the ‘pregnant’ and the ‘non-pregnant’ horns
(Figs 13 and 14). The chorioallantois, in this period, by
progressive increase of tissue mass, loses its transpar-
ency (Figs 13 and 14); this phenomenon is specifically
caused by the cotyledons, which become bigger and
more numerous in a negative gradation from the foetal
zone to the tips of chorioallantois. Both tips end in thin
chorionic projections which degenerate physiologically
because they are not supplied by allantoic vasculature
from inside (Figs 13 and 14). Most allantoic vessels are
observed near the foetus and progressively become
thinner towards the gestational sac extremities – the
mesometrially oriented vessel bundle being the most
evident (Fig. 13). On day 70 p.i., the allantoic vascula-
ture ‘stains’ the chorionic villi so distinctly that the
substructure of the largest cotyledons near the foetus
becomes macroscopically visible (Fig. 15). This phe-
nomenon can not only be observed in chorioallantoic
cotyledons but also in amniochorionic cotyledons
(Fig. 15). The amnion does not change in size in relation
to the foetus from days 50 to 70 p.i. (Figs 13–15). The
umbilical cord is now as long as the hind leg (Fig. 15),
with the umbilical vessels (allantoic arteries and veins)
being evident. A very small (involutive) yolk sac is
observable at this time, being located near the cranial
umbilical artery ramification (Fig. 12).
768 AC Assis Neto, FTV Pereira, TC Santos, CE Ambrosio, R Leiser and MA Miglino

Fig. 13. Foetus 4.2 cm CR,

53 days with drawing of the
sausage-likely chorioallantois sac
with extension of full length
(75 cm) inside the uterus. The
constriction of allantochorion is
corresponding to the corpus uteri
(arrows) and marks, on one side,
the larger part of this sac with the
amnion vesicle surrounding the
foetus in the so-called pregnant
uterine horn and, on the other, the
smaller and shorter part in the
unpregnant horn. Both parts end in
* thin chorionic projections which
degenerate because they have not
been supplied by allantoic vessels
from inside (*). Cotyledons best
* are developed on the zone of
chorioallantois near the foetus,
whereas towards the tips of gesta-
tions sac, they yet are gradually
smaller in number and size. Note
the distinct, mainly mesometrially
located allantoic vasculature not
yet fully reaching to the allanto-
chorionic tips. Scale bar 2 cm

AMC

ALC

Fig. 15. Foetus 10 cm CR, 70 days. Two bundles of blood vessels

from each side of chorioallantois extremes (arrows) meet in the
umbilical cord (triangle) to form the umbilical arteries and vein. The
distinct cotyledons show the substructure of villosity. ALC, chorio-
allantois with corresponding cotyledons; AMC, amniochorion with
corresponding cotyledons. Scale bar 2 cm

Fig. 14. Foetus 7.9 cm CR, 60 days. The chorioallantois sac by its
increased tissue mass is less transparent by light than before. Easily to
see are only the allantoic vessels along the mesometrial line of sac as 105 16
Chorioallantois and amnion length (cm)

Chorioallantois Amnion Yolk sac

well as caruncles, which manually can be separated from the uterine
caruncles. Remnant of chorionic sac (arrow). Scale bar 2 cm 90 14

12
Yolk sac length (cm)

75
Morphometrical evaluation of embryo–foetal membranes 10
related to external characteristics of corresponding 60
embryos and foetuses 8
45
Lengths of chorioallantois and amnion 6
30
The length of chorioallantois reveals slow and gradual 4
development within evolution of gestational period from 15 2
days 20 to 70 p.i. (Fig. 16). It is about 40 to 75 cm
during this time. However, these lengths are discretely 0 0
by slower growth from days 50 to 60 of gestation, in 20 – 25 25 – 30 30 – 40 40 – 50 50 – 60 60 – 70
Days p.i. of pregnancy
relation to that one in the subsequent period from days
60 to 70. The length of amnion shows gradual growth Fig. 16. Lengths (cm) of placental membranes in bovine placenta from
until 40 to 50 days of gestation. However, there is a 20 to 70 days of pregnancy

 2009 Blackwell Verlag GmbH

Macroscopy of Bovine Embryo ⁄ Foetus and Membranes
tendency of faster growth from days 40 to 70 p.i.
(Figs 8, 9 and 16).
Length of yolk sac
The bovine yolk sac, which is about 9 cm on day 25 p.i.,
decreases in total length permanently during this study.
In most cases, the yolk sac disappears completely from
days 50 to 70 p.i.; however, in some cases, vestiges of the
central part of this sac can be observed during this time
(Figs 12 and 16; Table 3).
Number of cotyledons
The cotyledons are not macroscopically observable
until about day 37 of bovine gestation. Initially, from
37 to 40 days p.i., a maximum of 20 cotyledons are
present on the chorionic surface in the embryo. From
day 40 to 50 p.i., the number of cotyledons triples. In
the subsequent duration, i.e. up to day 70 p.i., the
number increases gradually, averaging about 80
(Fig. 17).
Greatest length and weight of conceptuses
The GL of conceptus specifically increases from 20 to 25
and from 60 to 70 days of gestation, in total, from 0.3 to
70 cm. The mean weight of the conceptus is 0.25 g on
day 20 and increases relatively slowly until 40 days up to
0.77 g. Then, the weight progressively increases dis-
tinctly up to 5.90 g at day 60. Finally, there is a steep
increase up to 14.53 g on day 70 p.i. (Fig. 18).
100
80
Number of cotyledons
60
40
20
0
30 – 40 40 – 50 50 – 60
Days p.i. of pregnancy
Fig. 17. Numbers of cotyledons on bovine placenta from 30 to
70 days of pregnancy
 2009 Blackwell Verlag GmbH
769
80 16
Greatest length of conceptus (mm)
Greatest length (mm)
70 14
Weight of conceptus (mm)
Weight (g)
60 12
50 10
40 8
30 6
20 4
10 2
0 0
20 – 25 25 – 30 30 – 40 40 – 50 50 – 60 60 – 70
Days p.i. of pregnancy
Fig. 18. Greatest lengths ( ) and weights (¤) of bovine embryos
compared in periods during 25 to 70 days of pregnancy
Discussion
Why is an ‘embryo ⁄ foetal age-scale’ needed in early
bovine development?
The literature on the intrauterine development of the
conceptus and its external membranes in bovine species is
rather voluminous but intriguing with respect to the exact
timing of the age of the embryo ⁄ foetus. The only way to
obtain this information on the age of the conceptus is by
insemination under time control by natural mating or
artificial means. There are rather few studies of this
information as shown in Table 1, which helps in getting
an applicable scale. The age of the conceptus is deter-
mined by the many typical morphological characteristics
that it shows. Part of the morphological studies presented
here by the investigation of the bovine conceptus and
related membranes during day 20 to 70 of pregnancy may
enhance the value of this age-scale.
Two facts make this scale so important: (i) of late, the
cow is being extensively used as an ‘experimental
animal’ for studying new techniques of reproduction
like in vitro fertilization (Farin and Farin 1995), embryo
transfer (Bertolini et al. 2002) and cloning (Smith and
Murphy 2004). (ii) The cow is an expensive experimental
animal, excluding a reasonably great number of animals
of exactly known pregnancy age needed for those
experiments. However, there is a large and relatively
easily available pool of pregnant cows from slaughter-
houses, the pregnancy age of which is unknown. This
pool was used for deriving the embryo ⁄ foetal age-scale
of the study presented here, which is confirmed by
relevant literature and complemented by our data
relating to exterior macroscopical morphology of con-
ceptuses.
How precise can the embryo–foetal age-scale be in early
bovine development of conceptus?
It is obvious that even if we carefully control and select
statistically improved data from the literature and from
results of this study, the embryo ⁄ foetus age-scale pro-
posed here cannot be used without including consider-
60 –70 able variability of the macroscopical appearance. This
variability is mostly due to biological reasons. Winters
et al. (1942) state that the actual time of fertilization or
ovulation cannot be exactly determined in bovine
species. In addition, two types of factors influence the
770 AC Assis Neto, FTV Pereira, TC Santos, CE Ambrosio, R Leiser and MA Miglino
growth of an embryo: (i) embryo-related factors like
individuality or genetic constitution (Evans and Sack
1973), breed (McGeady et al. 2006) and sex; and (ii)
mother-related factors like age, nutrition and uterine
environment. The growth rate can also vary enor-
mously, relating to just one specific phenomenon only,
e.g., the length of the chorionic sac during the elonga-
tion phase (Betteridge et al. 1980; Betteridge and
Fléchon 1988; Wrobel and Suess 1998). Other param-
eters are more ‘stable’ and related to age, particularly
those taken from the embryo ⁄ foetus exterior, like
neurulation, somites (McGeady et al. 2006), C-form of
embryo body, branchial arcs, pigmentation of the eye
and legbud formation. The weight of the conceptus is
helpful; however, it is critical if it is not being measured
under similar circumstances. The most used age crite-
rion is the length of embryo ⁄ foetus. After Winters et al.
(1942), we preferred the ‘greatest length’ (greatest
diameter of conceptus, head and rump together) to the
crown–rump length (Noden and Lahunta 1985; McGe-
ady et al. 2006), because the former is more stable when
comparing the different ages, particularly during the
C-form period of the embryo. Even with the uncertain-
ties of the embryo ⁄ foetus age-scale mentioned here, this
scale can be a very valuable tool in experimental work
with early bovine conceptuses not only from cows
controlled by pregnancy time but also from uncon-
trolled pregnant slaughterhouse animals.
Macroscopical morphology of early bovine conceptus–
maternal interrelationship – no more an enigma?
Why did we start the age-scale with about day 8 p.i.?
Before this time, the bovine embryos can be harvested
relatively easily, be it by superovulation with numer-
ously enhanced germs or by in vitro fertilization,
followed by flushing from the uterus (Maddox-Hyttel
et al. 2003). But, after this time, which represents the
logarithmic elongation growth phase, the conceptuses
show striking variability in size (Betteridge et al. 1980)
with, e.g., the length of the chorion extending two-thirds
of the pregnant uterine horn on day 14 p.i. (Winters
et al. 1942). Chorion of this length is an obstacle for
flushing out the embryo from the uterus, because of the
mechanical difficulties in handling the blastocyst (Bette-
ridge et al. 1980). Therefore, after about day 15 p.i.,
experiments which require morphological visualization
of the conceptus or tissue probes from it can be
performed only on the uterus from the slaughtered cow.
In additional to the mechanical problems of handling
the conceptus, giant cells first appear in the trophoblast
of chorion of day 16 to 18 p.i. embryos (Greenstein et al.
1958), which is a distinct sign that preparations for
implanting the chorion to the uterine epithelium take
place, as interpreted by Leiser (1975) as the ‘precontact
phase’. Implantation (Leiser 1975) then, at days 18 ⁄ 19
p.i., develops a corresponding apposition between tro-
phoblast and uterine epithelial microvilli which, 2 days
later, interdigitate each other, characterizing the adhe-
sion stage of implantation. This is the firmest morpho-
logical materno-embryonal interrelationship of
implantation possible in bovine species. While the
chorionic sac reaches the tips of both uterine horns by
elongation, this three-phase implantation process, being
observable only by transmission electron microscopy,
extends up to day 27 p.i. from the embryo zone along
the surface of the chorion to the extremes. When
arriving at this process, the implantation is complete.
With the exception of the embryo and its surrounding
amnion, the embryonal membranes are barely visible in
the open uterus from day 20 to about 27 p.i. (Leiser
1975; this study). However, the chorion, when fixed with
formaldehyde becomes wrinkled and opaquely trans-
parent, with a relatively weak implantative attachment
to the uterus, and can be detached from it carefully, and
the whole conceptus taken off from this organ (Fig. 2;
Leiser 1975).
On about day 30 p.i., the conceptus is typically
sausage-like (Melton et al. 1951; this study) and the
chorion, evidently under pressure, tends to slough out
from the cut uterus, by this obviously detaching the
implantation-related embryo–maternal connection. This
pressure is obviously built up by the allantoic fluid,
which is produced in a fairly large amount in a relatively
short time (see Figs 3 and 4 in Melton et al. 1951); it
enables the chorion to cling narrowly to the endometrial
caruncles and to form chorionic villi from day 31
(Melton et al. 1951; Greenstein et al. 1958) up to 33 p.i.,
which are only microscopically detectable at first, clearly
penetrating the caruncular septa (Melton et al. 1951;
King et al. 1979). Leiser et al. (1998) showed that cows
exhibit a relatively firm placental anchorage based on
the elaborate villous cotyledons of the placentomes. This
placentome development process, which macroscopi-
cally becomes first visible at about day 37 p.i. when the
allantois vasculature of the chorionic villi is a substantial
part of them, spreads out from the embryo zone to the
tips of gestation sac up to day 50 p.i. This totals to a
maximum of 40 placentomes, but this materno-foetal
connection cannot prevent the gestational sac from
forcing its way out from the uterus. On about day 70
p.i.; however, the placentome develops firm enough that
the conceptus stays inside the uterus after being cut
open, a fact confirmed by Chang (1952). This connec-
tion, which can be detached quite easily manually, was
incorrectly taken to be implantation in the bovine
(Kingman 1948; overview by Leiser 1975). The bovine
placentomes of the concepto-maternal attachment
become improved by continuous growth in number –
up to day 70 p.i. about 80 – in size, and in progressively
more mushroom-like form accompanied by interior
remodelling, as histologically investigated by Bjoerkman
(1954, 1969).
What is macroscopically visible of the development of
earliest bovine blood vasculature?
The first real blood circulation in the bovine is linked to
the development of the yolk sac (see below). The blood
vessels of the yolk sac are not directly visible macro-
scopically but the specific red colouration of the blood
or erythrocytes therein is evident. The intensity of this
colouration corroborates with the activity of erythro-
poiesis which probably culminates between days 25 and
30 p.i. (Figs 3 and 4), a fact, which should be improved
upon histologically, however.
 2009 Blackwell Verlag GmbH
Macroscopy of Bovine Embryo ⁄ Foetus and Membranes
The second and definitive blood vascular system is
generated by the allantois, which for the first time can be
observed at days 20 to 21 p.i. as a bud (Fléchon 1978;
Maddox-Hyttel et al. 2003), and is more prominent
1 day later (Zietzschmann 1924; Winters et al. 1942)
with a distinct allantoic stalk (Wrobel and Suess 1998).
The first macroscopically visible allantoic vasculature,
sprouting peripherally from the ventral side of the
embryo and following a mesometrially oriented line
(Figs 3 and 4), appears at day 26 p.i., as confirmed by
Greenstein et al. (1958). These authors show histolog-
ically that capillaries are situated along the trophoblast
of the chorion 3 days later, thus proving that a fusion of
the vessel-bearing allantois and the chorion has already
taken place. This fusion was also confirmed by Chang
(1952) and Schlafer et al. (2000) on day 30 p.i. However,
by macroscopy, this fusion cannot be ascertained to
have taken place before day 36 p.i. That is, this becomes
visible when the part of allantois showing allantoic
vessels sticks to the chorion and both of them can be
separated by manipulation. This fusion, on day 36 p.i.,
must have become distinctly firmer than before, because
for the first time cotyledons on the chorion become
macroscopically evident, a fact which is explainable by a
distinct amount of blood in the cotyledons, ‘staining’
them distinctly red (Fig. 7). This phenomenon, after
about day 60 p.i., is striking; the roundish cotyledons
which are contrasting because of their red colour have
increased to an average diameter of about 2 cm in which
even the substructure of chorionic villi is visible because
of their vascular blood content (Fig. 15). On day 45 p.i.,
a very faint allantoic vasculature is macroscopically
evident on the amnion, indicating the fact that an
allantamnion has developed. As concerns the fusion of
the amnion with the chorion, it is not yet evident if
amniochorionic cotyledons, being evident at around day
70 p.i. (see also Schlafer et al. 2000), are linked with
such a process. Because no fusion by mesenchyme layer
has been observed so far, the amniochorionic cotyledons
are ‘just’ allantochorionic cotyledons which have been
developed on the location where the fusion between
amnion and chorion will be established later, an
observation definitely stated on day 98 p.i. by Weeth
and Herman (1952).
The development of the allantochorion from the
embryo ⁄ foetus zone to its extremes is macroscopically
well traced out by its vascularization. This starts from
the main vessels of the umbilicus, which is clearly visible
on day 33 p.i. From this day on, vessels become smaller
and less densely ramified by branching (Fig. 13), but
gradually grow towards the end of the gestation sac by
distinctly reaching it up to day 70 p.i. of pregnancy as
shown in our study. However, this vascular system with
inclusion of the cotyledons is distinctly better developed
in the ‘pregnant’ uterine horn than in the non-pregnant
horn. Both gestation sacs end abruptly, obviously filling
the uterine horns completely. This is explainable by the
fact that the allantois, which has penetrated the chori-
onic sac as far as possible, to widen for a firm contact to
the uterus and to form the placentomes for attachment
and nutrition of the conceptus. The chorion not used
that way is visible as degenerating necrotic tips in
Fig. 13.
 2009 Blackwell Verlag GmbH
771
The yolk sac in the bovine? Yes, it is macroscopically
evident!
The yolk sac in the bovine develops from days 18 to 23
p.i., when the embryo converts from a flat trilaminar disc
into a cylindrical body (Wrobel and Suess 1998). On day
22 p.i., it is composed of three parts: a broad vitelline
connection to the intestine of the conceptus, a centre and
two projections, of which the projections reach into the
chorionic ends (Rüesse et al. 1992). We found that at
about day 25 p.i., the yolk sac is macroscopically evident
by the vitelline duct, a centre and two projections (Figs 3
and 4), which has a total length of about 10 cm. This
dominance may explain the importance of the yolk sac,
particularly during this time, as an organ for generating
the primordial germ cells (Jost and Prepin 1966; Wrobel
and Suess 1998; Kritzenberger and Wrobel 2004) and
for erythropoiesis, i.e. development of blood islands
into the vitelline circulatory blood system, as a first
transport system for metabolites between the mother
and the embryo or between the periphery and centre of
the conceptus (Rüesse et al. 1992; Kritzenberger and
Wrobel 2004). On day 30 p.i. of pregnancy, the yolk sac
is smaller than before, about 6 cm in length, because the
tips of projections degenerate (32 day p.i.; Zietzschmann
1924). This is a regressive process, and the yolk sac can
be macroscopically found on day 55 p.i., at the latest, as
a spindle-shaped rudiment being barely visible and
located cranially of the umbilical cord. In our study,
we described for the first time in B. indicus, the transition
period of the choriovitelline placenta (McGeady et al.
2006) until it was replaced by the chorioallantoic
placenta.
Conclusion
In conclusion, all the data collected in this study were
compared extensively with the corresponding data of the
literature. This resulted in an ‘embryo ⁄ foetal age-scale’
which, combined with data from this study, has been
extended, covering the time of 8 to 70 days p.i. By using
this age-scale of early bovine intrauterine development,
macroscopic identification of the stages of embryonic
development becomes possible even when using embryos
from slaughtered cows without distinct knowledge of
insemination or fertilization time. This distinctly facili-
tates research into the cow, which is fast becoming ‘an
experimental animal of choice’ for testing new techniques
like in vitro fertilization, embryo transfer and cloning.
Acknowledgement
This study was supported by Fundação de Amparo a Pesquisa do
Estado de São Paulo (FAPESP), Process number 02 ⁄ 00233-0 and
06 ⁄ 53009-1 and also to Coordenação de Aperfeiçoamento de Pessoal
de Nı́vel Superior (CAPES). The authors thanks Dr. Ricardo da
Fonseca for help with the statistical analysis.
Author contributions
Dr Antonio Chaves de Assis Neto and Dr Maria Angelica Miglino
chiefly carried out the research. Antonio C.A. Neto, Flávia T.V.
Pereira, Tatiana C. Santos and Carlos E. Ambrosio harvested and
processed material; Antonio C.A. Neto, Prof Rudolf Leiser and Prof
Miglino analysed data and made descriptions of results.
772 AC Assis Neto, FTV Pereira, TC Santos, CE Ambrosio, R Leiser and MA Miglino

References a close relationships between hematopoietic stem cells and

primordial germ cells. Histochem Cell Biol 121, 273–289.
Bertolini M, Mason JB, Beam SW, Carneiro GF, Sween ML,
Leiser R, 1975: Development of contact between trophoblast
Kominek DJ, Moyer AL, Famula TR, Sainz RD, Anderson
and uterine epithelium during the early stages of implanta-
GB, 2002: Morphology and morphometry of in vivo- and in
tion in the cow. Anat Histol Embryol 4, 63–86.
vitro-produced bovine concepti from early pregnancy to
Leiser R, Pfarrer C, Abd-Elnaeim M, Dantzer V, 1998: Feto-
term and association with high birth weights. Theriogenol-
maternal anchoring in epitheliochorial and endotheliocho-
ogy 58, 973–994.
rial placental types by studied histology and microvascular
Betteridge KJ, Fléchon J-E, 1988: The anatomy and physiol-
corrosions cast. Troph Res 12, 21–39.
ogy of preattachment bovine embryos. Theriogenology 29,
Maddox-Hyttel P, Alexopoulos NI, Vajta G, Lewis I, Rogers
155–187.
P, Cann L, Callesen A, Tveden-Nyborg P, Trounson A,
Betteridge KJ, Eaglesome MD, Randall GC, Mitchell D, 1980:
2003: Immunohistochemical and ultrastructural character-
Collection, description and transfer of embryos from cattle
ization of the initial post-hatching development of bovine
10–16 days after oestrus. J Reprod Fertil 59, 205–216.
embryos. Reproduction 125, 607–623.
Bjoerkman N, 1954: Morphological and histochemical studies
McGeady TA, Quinn PJ, Fitzpatrick EP, Ryan MT, 2006:
on the bovine placenta. Acta Anat Suppl 22, 1–91.
Veterinary Embryology. Blackwell Publishing, Oxford.
Bjoerkman N, 1969: Light and electron microscopic studies on
Melton AA, Berry RO, Butler OD, 1951: The interval between
cellular alterations in the normal bovine placentome. Anat
the time of ovulation and attachment of the bovine embryo.
Rec 163, 17–30.
J Anim Sci 10, 993–1005.
Cavalcante FA, Filho RM, Campelo CC, Lobo RNB, Martins
Newcomb R, Christie WB, Rowson LEA, 1978: Nonsurgical
GA, 2001: Gestation length of Nellore cattle in the Oriental
recovery of bovine embryos. Vet Rec 102, 414–417.
Amazonia. Rev Bras Zootec 30, 1451–1455.
Noden DM, Lahunta A, 1985: The Embryology of Domestic
Chang MC, 1952: Development of bovine blastocyst with a
Animals. William & Wilkins, Baltimore, MD.
note on implantation. Anat Rec 113, 143–161.
Paschal JC, Sanders JO, Kerr JL, 1991: Calving and weaning
Chenoweth PJ, 1994: Aspects of reproduction in females Bos
characteristics of Angus-, Gray Brahman-, Gir-, Indu-
indicus cattle: a review. Aust Vet J 71, 422–426.
Brazil-, Nellore-, and Red Brahman-sired F1 calves. J Anim
Evans HE, Sack WO, 1973: Prenatal development of domestic
Sci 69, 2395–2402.
and laboratory mammals: growth curves, external features
R Development Core Team, 2006: R: A Language and
and selected references. Anat Histol Embryol 2, 11–45.
Environment for Statistical Computing. R Foundation for
Farin PW, Farin CE, 1995: Transfer of bovine embryos
Statistical Computing, Vienna. ISBN 3-900051-07-0; http://
produced in vivo or in vitro: survival and fetal development.
www.R-project.org.
Biol Reprod 52, 676–682.
Rüesse I, Sinowatz F, Richter L, Lehmann M, Schallenberger
Fléchon J-E, 1978: Morphological aspects of embryonic disc at
E, 1992: Die Entwicklung des Dottersackes beim Wie-
the time of its appearance in the blastocyst of farm animals.
derkäuer (Schaf und Rind). Anat Histol Embryol 21, 324–
Scan Electr Microsc 2, 541–548.
347.
Greenstein JS, Murray RW, Foley RC, 1958: Observations on
Schlafer DH, Fisher PJ, Davies CJ, 2000: The bovine placenta
the morphogenesis and histochemistry of the bovine pla-
before and after birth: placental development and function
centa between 16 and 33 days of gestation. Anat Rec 132,
in health and disease. Anim Reprod Sci. 60–61, 145–160.
321–341.
Smith LC, Murphy BD, 2004: Genetic and epigenetic aspects
Hamilton WJ, Laing JA, 1946: Development of the egg of the
of cloning and potential effects of offspring of cloned
cow up to the stage of blastocyst formation. J Anat 80, 194–
mammals. Clon Stem Cells 6, 126–132.
209.
Weeth HJ, Herman HA, 1952: A histological and histochem-
Hill JR, Burghardt RC, Jones K, Long CR, Looney CR, Shin
ical study of the bovine oviducts, uterus and placenta. Univ
T, Spencer TE, Thompson JA, Winger QA, Westhusin ME,
Missouri Coll Agric Exp Sta Res Bull 501, 1–54.
2000: Evidence for placental abnormality as the major cause
Winters LM, Green WW, Comstock RE, 1942: Prenatal
of mortality in first-trimester somatic cell cloned bovine
development of the bovine. Univ Agric Exp Sta Minnesota
fetuses. Biol Reprod 63, 1787–1794.
Tech Bull 151, 3–50.
Jost A, Prepin J, 1966: Données sur la migration des cellules
Wooding FBP, Flint APF, 1994: Placentation. In: Lamming
germinales primordiales du foetus de veau. Arch Anat
GE (ed.), Marshall’s Physiology of Reproduction. Chapman
Microsc 55, 161–186.
and Hall, London.
King GJ, Atkinson BA, Robertson HA, 1979: Development of
Wrobel K-H, Suess F, 1998: Identification and temporospatial
the bovine placentome during the second month of gesta-
distribution of bovine primordial germ cells prior to gonadal
tion. J Reprod Fertil 55, 173–180.
sexual differentiation. Anat Embryol 197, 451–467.
King GJ, Atkinson BA, Robertson HA, 1980: Development of
Zietzschmann O, 1924: Lehrbuch der Entwicklungsgeschichte
the bovine placentome from days 20 to 29 of gestation. J
des Haustiere. Richard Schoetz, Berlin.
Reprod Fertil 59, 95–100.
Kingman HE, 1948: The placentome in the cow. Am J Vet Res Submitted: 02 Oct 2008
9, 125–130.
Kritzenberger M, Wrobel K-H, 2004: Histochemical in situ Author’s address (for correspondence): A C Assis Neto, Campus
identification of bovine embryonic blood cells reveals Experimental de Dracena, São Paulo State University, 17900-000,
differences to the adult hematopoietic system and suggests Dracena, São Paulo, Brazil. E-mail: antonioassis@dracena.unesp.br

 2009 Blackwell Verlag GmbH