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Ultrastructural characterization of fresh and cryopreserved

in vivo produced ovine embryos

E.M.V. Bettencourt a,*, C.M. Bettencourt b, J.N.C.E. Silva c, P. Ferreira d,C.P. de Matos b, E. Oliveira e, R.J. Romao f, A. Rocha d, M. Sousa e

a Department of Veterinary and Institute of Mediterranean Agricultural Sciences (ICAM), University of Evora, 7000-092 Evora, Portugalb Regional Agriculture Direction of Alentejo (DRAAL), Herdade da Abobada, V.N.S. Bento, Portugal

c Ex-Division for Animal Selection and Reproduction (DSRA), and Veterinary General Direction (DGV), Amadora, Portugald Campus Agrario de Vairao, University of Porto, Portugal

e ICBAS-Lab Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Portugalf Department of Zootechny, and Institute of Mediterranean Agricultural Sciences (ICAM), University of Evora, Evora, Portugal

Received 23 May 2008; received in revised form 22 September 2008; accepted 27 October 2008

Abstract

Controlled slow freezing and vitrification have been successfully used for ovine embryo cryopreservation. Selection of embryos

for transfer is based on stereomicroscopical embryo scoring after thawing, but the subjectivity inherent to this selection step has

been demonstrated by ultrastructural studies of controlled slow frozen, in vivo produced ovine morulae and blastocysts. These

studies have shown that certain abnormalities remain undetected by stereomicroscopy only. In the present study, using ovine in vivo

produced morulae and blastocysts, we have studied the ultrastructural alterations induced by open pulled straw vitrification (OPS)

and controlled slow freezing, compared stereomicroscopical embryo scoring with light microscopy evaluation of embryo’s semithin

sections, and related the ultrastructural cellular damage with the embryo classification by stereomicroscopical embryo scoring of

embryos’ and semithin section evaluation by light microscopy. The ultrastructural lesions found for OPS-vitrified and controlled

slow frozen embryos were similar, independently of embryo stage. A significant higher number of grade 3 embryos was found at

stereomicroscopical scoring after controlled slow freezing (P = 0.02), and a significant higher number of grade 3 blastocysts was

found at semithin sectioning after OPS vitrification (P = 0.037). The extension of ultrastructural damage, especially of

mitochondria and cytoskeleton, was related to the semithin classification but not to stereomicroscopical scoring at thawing. This

suggests that semithin scoring is a useful tool for predicting ultrastructural lesions and new improvements in cryopreservation and

thawing methods of ovine embryos are still warranted, including in the case of blastocysts cryopreserved by OPS vitrification.

# 2008 Elsevier Inc. All rights reserved.

Keywords: Cryopreservation; OPS vitrification; Ovine embryo morphology; Ultrastructure

www.theriojournal.com

Available online at www.sciencedirect.com

Theriogenology xxx (2008) xxx–xxx

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produced ovine embryos. Theriogenology (2008), doi:10.1016/j.t

* Corresponding author. Tel.: +351 266760809;

fax: +351 266760944.

E-mail address: emvb@uevora.pt (E.M.V. Bettencourt).

0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2008.10.019

1. Introduction

In order to increase the genetic gain resulting from

selection, efficient cryopreservation protocols are

required to allow delayed transfer of embryos and for

incrementing the use of multiple ovulation and embryo

transfer techniques [1,2]. Cryopreservation of embryos

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from many mammalian species have seen a widespread

use in reproductive research and in animal breeding in

recent years [2–4], but the technique has a much lower

utilization in sheep than, for example, in cattle [5]. This

fact may be related to the relatively high cost of the

technique in sheep when compared to the value of the

animals. Reducing costs at any stage of embryo

production and cryopreservation, including through

an increase in the efficiency of cryopreservation

methods, is likely to augment multiple ovulation and

embryo transfer techniques utilization in the ovine

species [1,4].

Controlled slow freezing and vitrification have been

the two major techniques used for embryo cryopre-

servation [4,6]. Controlled slow freezing was intro-

duced firstly and remains the mostly common technique

used, both with domestic animal and human embryos

[6]. However, slow-freezing requires expensive biolo-

gical freezers and is time consuming. The use of

ultrarapid techniques, such as vitrification, has reduced

part of the costs, since it does not require any special

equipment and is, therefore, well adapted to routine

field use [1]. The open pulled straw (OPS) method was

later developed [7] as a modification of embryo

vitrification, allowing increased cooling rates without

affecting embryo survival.

Controlled slow freezing and OPS have been used for

the cryopreservation of ovine morulae and blastocysts

produced in vivo [1,8–11] and in vitro [10,11]. The loss

of viability associated with freezing can be influenced

by the type and concentration of the cryoprotectant

used, the freezing protocol, the species and genotype of

the animal, the developmental stage of the embryos and

the type (in vivo vs in vitro) of embryo production

[2,3,6]. Comparisons between the different techniques

have relied on lambing rates after embryo transfer.

However, selection of embryos for transfer is based on

the stereomicroscopical evaluation of embryo morphol-

ogy after thawing [12] in accordance to the guidelines of

the International Embryo Transfer Society [13]. The

subjectivity inherent to this selection step has been

demonstrated by ultrastructural studies of vitrified in

vitro and in vivo produced bovine blastocysts [14], and

of controlled slow frozen in vivo produced ovine

morulae and blastocysts [15], which have shown that

certain abnormalities remain undetected by stereomi-

croscopy.

The ultrastructure of fresh and controlled slow frozen

in vivo produced ovine morulae and blastocysts has

been studied [15,16], but no reports have described the

ultrastructural features of OPS vitrified embryos nor

compared the two methods. The aims of the current

Please cite this article in press as: Bettencourt EMV, et al. Ultras

produced ovine embryos. Theriogenology (2008), doi:10.1016/j.t

study were: (a) to investigate the ultrastructural

alterations induced by OPS vitrification of in vivo

produced ovine embryos at the morula and blastocyst

stages, (b) compare streomicroscopical embryo scoring

with light microscopy scoring of semithin sections, (c)

relate ultrastructural cellular damage with embryo

classification by stereomicroscopical scoring and light

microscopy semithin evaluation.

2. Materials and methods

2.1. Embryo production and embryo score

In vivo produced embryos (n = 51) were obtained

from Portuguese Black Merino ewes, superovulated

during spring. Donors were synchronized with

intravaginal sponges containing 40 mg flurogestone

acetate (Chronogest, Intervet Laboratories, Boxmeer,

Holland) for 12 days. Starting on day 9 of sponge

treatment and for 4 consecutive days, ewes were

treated (im) with 1.25 ml oFSH (Ovagen, Immuno-

logical Products Ltd, Aukland, New Zealand), twice a

day time (9:00 and 21:00). Sponges were removed at

the time of the 7th oFSH administration. Laparo-

scopic intrauterine insemination was performed with

fresh diluted semen in all ewes, 48 h after sponge

removal, as described [17]. A minimum of 50 � 106

motile spermatozoa was placed in each uterine horn.

Additionally, ewes were hand mated at 36 and 48 h

after sponge withdrawal and left with rams for 4–6 h

at a ewe:ram ratio of 3:1. Embryos were recovered by

abdominal laparotomy, under general anesthesia, on

day 6 or 7 after sponge removal. Each uterine horn

was flushed using a Foley catheter as described [18].

The collection medium (40 ml for each uterine horn)

was PBS containing 2% BSA.

Stereomicroscopical scoring of embryos was per-

formed based on morphological criteria and develop-

mental stage in accordance to International Embryo

Transfer Society guidelines [13]. Only fresh embryos of

grade 1 or grade 2 were selected for freezing. Fresh

embryos classified as grade 1 or 2 (n = 11) were directly

processed for transmission electron microscopy.

Collected grade 1 and 2 embryos at the morula and

blastocyst stages were immediately fixed or processed

for controlled slow freezing and OPS vitrification.

Frozen embryos were stereomicroscopical scored 1 h

after thawing and rescored at semithin sectioning. In the

smithin sections, embryos were scored as grade 1

(excellent or good) when presenting a symmetrical and

spherical cell mass with�85% of intact cells (<25% of

degenerated cells/cell fragments), with individual cells

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Fig. 1. OPS vitrified in vivo produced ovine embryos fixed 1 h after thawing. Semithin sections of morulae (A–C) and blastocysts (D–F). Embryo

grade 1 (A,D), grade 2 (B,E) and grade 3 (D,F). Zona pellucida (ZP), trophectoderm (TF), inner cell mass (ICM), blastocelic cavity (bc), degenerated

cells (D).

showing an uniform size, color and density; grade 2

embryos (fair) had a cell mass with�50% of intact cells

(25–50% of degenerated cells/cell fragments), with

individual cells presenting moderate irregularities in

size, color and density; and grade 3 embryos (poor) had

a cell mass with �25% of intact cells (>50% of

degenerated cells/cell fragments), with individual cells

presenting major irregularities in size, color and density

(Fig. 1).

2.2. Embryo freezing

Embryos stereomicroscopically scored as grade 1

and 2 (n = 40) were randomly distributed between

assigned to the two cryopreservation protocols, while

being kept in liquid nitrogen (�196 8C) for 3–6 months.

Embryo handling was performed at room temperature.

Controlled slow freezing was performed as described

[19] with modifications. Embryos were immersed for

5 min in a cryopreservation solution made of 0.4% BSA

in PBS with 1.5 M ethylene glycol (EG) added in one

step. Two embryos were then loaded into the middle

portion of each 90 mm straw (PBS, air bubble, PBS, air

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produced ovine embryos. Theriogenology (2008), doi:10.1016/j.t

bubble, cryopreservation solution with embryos, air

bubble, 0.3 M sucrose). Straws were sealed with plastic

ploughs (quicklock) and placed vertically in the cooling

chamber of a programmable freezer (Bio-Cool III, FTS

Systems Inc, Stone Ridge, NY), kept at�6 8C for 3 min

and then subjected to manual seeding. After 5 min at

�6 8C, embryos were cooled at 0.5 8C/min until

reaching �35 8C, kept for 15 min at �35 8C, and then

rapidly immersed in liquid nitrogen. For thawing,

straws were left 10 s in air and then for 10 s in a 35 8Cwater bath. After thawing, embryos were left for 5 min

in the content of the straw and then washed in holding

medium (4% BSA in PBS). Vitrification by the open-

pulled straw (OPS) method was conducted as described

[20]. Embryos were equilibrated for 5 min in holding

medium (HEPES-buffered TCM-199 medium supple-

mented with 20% of fetal calf serum), 3 min in dilution

medium (holding medium with 10% ethylene glycol

and 10% dimethyl sulfoxide) and 30 s in concentrated

medium (holding medium with 20% EG and 20%

DMSO). Embryos were loaded in the OPS through

capillarity, by placing the narrow end of the pulled straw

into the holding medium. The straws were then

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immediately submerged into liquid nitrogen. For

thawing, the end of the straw was directly immersed

in holding medium containing 0.25 M sucrose.

Embryos were left for 1 min in this medium, then

5 min in holding medium containing 0.15 M sucrose

and washed twice with holding medium.

2.3. Electron microscopy

Fresh (n = 11) and thawed embryos after controlled

slow freezing (n = 20) and OPS vitrification (n = 20)

were scored and then fixed, within 1 h after collection or

thawing, in Karnowsky (2 h, 4 8C), washed with 0.15 M

sodium cacodylate buffer, pH 7.3 (overnight, 4 8C),

post-fixed (1 h, 4 8C) in 1% OsO4 in buffer containing

0.8% hexanocyanoferrate potassium [K3Fe3+(CN)6],

and washed for 15 min in buffer. They were then

dehydrated through a graded series of ethanol (50, 70,

90, 2 � 100%; 30 min each), followed by propylene

oxide (15 min), impregnated with propylene oxide:epon

(3:1, 1 h; 1:1, overnight, 4 8C; 1:3, 4 h) and embedded

in Epon (7 h at room temperature, 3 days at 60 8C).

Sections were cut in a Leica ultramicrotome with a

Diatome knife. Semithin sections were stained with

aqueous azur II:methylene blue (1:1). Ultrathin sections

were collected on 200 mesh copper grids (Taab) and

stained with 3% aqueous uranyl acetate (20 min) and

Reynolds lead citrate (10 min). Sections were observed

in a transmission electron microscope JEOL 100CXII

operated at 60 kV.

Except where otherwise indicated, all chemicals

were obtained from Sigma Chemicals Company

(Barcelone, Spain).

2.4. Experimental design

Embryo scores were compared with 2-sided Fisher‘s

exact test (SPSS 15 for Windows).

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Table 1

Morphological classification of ovine embryos.

Cryopreservation method N Controlled Slow Freez

Embryo score (grade) 1 2

Thawing

Morulae 6 0 4

Blastocysts 14 8 2

Totals 20 8 6

Semithin sectioning

Morulae 6 0 6

Blastocysts 14 6 6

Totals 20 6 12

Numbers with lower case letters represent significant differences along lines,

3. Results

All fresh embryos were rescored at semithin

sectioning as grade 1 (2 embryos) or grade 2 (9

embryos). The majority of the thawed embryos were

classified either as grade 1 or grade 2 at both

evaluations, with the zona pellucida being intact in

all cases (Table 1). A significant higher number of grade

3 embryos was found at steromicroscopical scoring in

the controlled slow freezing method (P = 0.02), without

significant differences between morulae and blasto-

cysts. Although no significant differences were found in

the classification of the semithin sections (light

microscopy) between the two methods of cryopreserva-

tion, there was a significant higher number of grade 3

blastocysts in the OPS method (P = 0.037), as a higher

number of grade 3 embryos, especially of blastocysts

were found at the semithin sectioning classification than

at the stereomicroscopical classification, in the OPS

method (P = 0.003).

For the same grade at semithin scoring, no

differences (P > 0.05) were found regarding ultra-

structural characteristics between fresh, controlled slow

frozen and OPS vitrified embryos.

After OPS vitrification, morulae of grade 1 showed

an excellent preservation of the zona pellucida and of

both peripheral (Fig. 2A and B) and inner (Fig. 2C–F)

cells. No main differences were found between outer

and inner cells, with the exception of external microvilli

being present in outer cells (Fig. 2A and B). Cells were

characterized by the presence of intact intercellular

junctions, numerous mitochondria and light vesicles

(Fig. 2A–D). Mitochondria had abundant transversal

cristae and were surrounded by rough and smooth

endoplasmic reticulum cisternae (Fig. 2B,C, and F).

Light vesicles contained numerous small vesicles and

dense bodies, with coalescence between light vesicles

suggesting they represent maturing organelles (Fig. 2B–

tructural characterization of fresh and cryopreserved in vivo

heriogenology.2008.10.019

ing N OPS vitrification

3 1 2 3

2 14 8 6 0

4 6 0 6 0

6a 20 8 12 0bA

0 14 6 4 4

2a 6 0 2 4b

2 20 6 6 8B

and upper case letters represent significant differences along columns.

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produced ovine embryos. Theriogenology (2008), doi:10.1016/j.theriogenology.2008.10.019

Fig. 2. Ultrastructure of OPS vitrified in vivo produced ovine morulae fixed 1 h after thawing. Morulae were classified as grade 1 in semithin

sections. Outer (A,B) and inner (C–F) cells. Zona pellucida (ZP), microvilli (Mv), intercellular junctions (black arrows), mitochondria (m), light

vesicles (v), secondary lysosomes (LY), lipid droplets (L), Golgi complexes (G), smooth endoplasmic reticulum (white arrowheads), rough

endoplasmic reticulum (black arrowheads), nucleus (N), nuclear pore complexes (white open arrows), nucleolus (Nc), nucleolar fibrillar centers

(FC), nucleolar dense fibrillar component (DFC), nucleolar granular component (GC), polyribossomes (pr).

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produced ovine embryos. Theriogenology (2008), doi:10.1016/j.theriogenology.2008.10.019

Fig. 3. Ultrastructure of OPS vitrified in vivo produced ovine morulae fixed 1 h after thawing. Morulae were classified as grade 2 (A,B) and grade 3

(C–F) in semithin sections. Outer (A,C) and inner (B,D–F) cells. (A,B) Presence of extracellular cell debris (F), loss of microvilli and intercellular

junctions (black arrows), moderate swelling of the mitochondrial matrix (*), endoplasmic reticulum (white arrowheads) and light vesicles (v), and

occasional degenerated mitochondria (d). Lipid droplets (L). (C) Discontinuities of the cytoplasmic membrane (black open arrows), loss of

microvilli, disruption of intercellular junctions (black arrows), severe swelling of the mitochondrial matrix (**), mitochondria degeneration (d),

swelling of cytosolic areas (c) and swelling of the endoplasmic reticulum (white arrowheads). Zona pellucida (ZP), Golgi complex (G). (D) Large

aggregate of light vesicles (v), swollen endoplasmic reticulum (white arrowheads), and degenerated mitochondria (d). (E) Severe swelling of

mitochondria, with organelle rupture (**) and envelope degeneration (black open arrow). (F) Loss of intercellular junctions (black arrows), swelling

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produced ovine embryos. Theriogenology (2008), doi:10.1016/j.theriogenology.2008.10.019

Fig. 4. Ultrastructure of OPS vitrified in vivo produced ovine blastocysts fixed 1 h after thawing. Blastocysts were classified as grade 1 in semithin

sections. Blastocelic cavity (b), trophectoderm (A,B) and inner cell mass (C–F). Zona pellucida (ZP), microvilli (Mv), intercellular junctions (black

arrows), filaments associated with cell junctions (f), mitochondria (m), light vesicles (v), lipid droplets (L), Golgi complexes (G), smooth

endoplasmic reticulum (white arrowheads), rough endoplasmic reticulum (black arrowheads), nucleus (N), nucleolus (Nc), polyribossomes (pr).

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produced ovine embryos. Theriogenology (2008), doi:10.1016/j.theriogenology.2008.10.019

Fig. 5. Ultrastructure of OPS vitrified in vivo produced ovine blastocysts fixed 1 h after thawing. Blastocysts were classified as grade 2 (A,B) and

grade 3 (C–F) in semithin sections. Blastocelic cavity (b), trofectoderm (A,C,D) and inner cell mass (B,E,F). (A) Presence of cell debris (F),

cytoplasmic membrane discontinuities (black open arrow), decreased number and length of microvilli (Mv), moderate swelling of the endoplasmic

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D). In the remainder of the cytoplasm there were a few

Golgi complexes, secondary lysosomes, lipid droplets

and polyribosomes (Fig. 2A–D,F). The nucleus was

well defined and organized, with an intact nuclear

envelope, rich in nuclear pore complexes, and one or

more reticular nucleoli with evident fibrillar centers

surrounded by the dense fibrillar and granular compo-

nents (Fig. 2C–E).

At semithin scoring OPS vitrified morulae scored as

grade 2 also showed intact cellular structures in most of

the blastomeres, as described for grade 1 embryos.

However, there were several signs of cellular injury in

totally or partially detached cells. These ranged from

presence of cell debris, resulting from total cell lysis

(Fig. 3A), to decreased number and length of microvilli,

loss of intercellular junctions, and moderate swelling of

the mitochondrial matrix, endoplasmic reticulum and

light vesicles (Fig. 3A and B).

At semithin scoring, morulae vitrified by OPS and

classified as grade 3 presented additional degenerated

cells in both peripheral and internal cells, with badly

defined cellular limits, poor intracellular preservation,

disruption of the plasma membrane and presence of cell

debris, due to complete cell lysis. Less attained outer

cells showed discontinuities of the cytoplasmic mem-

brane, loss of microvilli, disruption of intercellular

junctions, severe swelling of the mitochondrial matrix,

degenerated mitochondria, focal swelling of cytosolic

regions and swelling of the endoplasmic reticulum

(Fig. 3C). Less attained inner cells showed vacuolar

degeneration, characterized by the presence of large

aggregates of light vesicles, swollen endoplasmic

reticulum and degenerated mitochondria (Fig. 3D),

while other cells presented severe swelling of mito-

chondria, with organelle rupture and envelope degen-

eration (Fig. 3E), loss of intercellular junctions,

swelling of the nucleoplasm and cytosol, disorganiza-

tion of the nucleolar structure, and cytoplasmic

membrane and nuclear envelope discontinuities

(Fig. 3F).

At semithin scoring, after OPS vitrification, blas-

tocysts of grade 1, showed an excellent preservation of

the zona pellucida, trophectoderm (Fig. 4A and B) and

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produced ovine embryos. Theriogenology (2008), doi:10.1016/j.t

reticulum (white arrowheads) and light vesicles (v). Zona pellucida (ZP), mito

loss of cristae (*), swelling of the endoplasmic reticulum (white arrowhead

extracellular cell debris (F). Nucleus (N), light vesicles (v), lipid droplets (L)

discontinuities (black open arrows), disruption of intercellular junctions (bla

light vesicles (v). Smooth endoplasmic reticulum (white arrowheads), rough

cytosol (c) and nucleoplasm (n), with disruption of intercellular junctions (bl

Degenerative cells, with loss of membrane limits (black arrows) and organel

open arrows), cytosol (c), degenerated mitochondria (d), light vesicles (v).

inner cell mass (Fig. 4C–F). No main differences were

found between cells of the trophectoderm and inner cell

mass, with the exception of the presence of external

microvilli in trophectoderm cells (Fig. 4A and B). Intact

intercellular junctions, consisting of tight junctions,

desmosomes and gap junctions, were observed in all

cells, including between the trophectoderm and inner

cell mass (Fig. 4A,C,D, and F). The cytoplasm

contained the same set of organelles previously found

in morulae, mitochondria with numerous transversal

cristae surrounded by cisternae of the smooth and rough

endoplasmic reticulum (Fig. 4B,C–F), more abundant

and well developed Golgi complexes (Fig. 4A and C),

light vesicles, lipid droplets, secondary lysosomes, and

polyribosomes (Fig. 4F). Cells displayed a large

euchromatic nucleus, with an intact nuclear envelope

rich in nuclear pore complexes and one or more reticular

nucleoli, with evident fibrillar centers surrounded by the

dense fibrillar and granular components (Fig. 4A,C, and

E).

At semithin scoring, OPS vitrified blastocysts scored

as grade 2 showed well preserved cellular structures but,

as described for grade 2 morulae, there were some signs

of cellular degeneration, such as extracellular cell

debris, decreased number of trophectoderm outer

microvilli, cytoplasmic membrane discontinuities,

swelling of light vesicles (Fig. 5A), mitochondria and

endoplasmic reticulum, and increased areas of the

cytoplasm without organelles (Fig. 5B).

At semithin scoring, blastocysts vitrified by OPS and

classified as grade 3 presented additional degenerated

cells in both trophectoderm and inner cell mass, with

badly defined cellular limits, poor intracellular preserva-

tion, disruption of the plasma membrane, nuclear

envelope and other organelle membranes, and presence

of cell debris due to complete cell lysis (Fig. 5F). Less

attained cells showed loss of microvilli (Fig. 5C),

disruption of intercellular junctions (Fig. 5D and E),

severe degenerative swelling of mitochondria (Fig. 5C

and D), discontinuities in the cytoplasmic membrane

(Fig. 5C and D) and nuclear envelope (Fig. 5E), as well as

swelling of the cytosol and nucleoplasm, with disorga-

nization of the nucleolar structure (Fig. 5E and F).

tructural characterization of fresh and cryopreserved in vivo

heriogenology.2008.10.019

chondria (m). (B) Moderate swelling of the mitochondrial matrix with

s), presence of large cytosolic areas devoid of organelles (c) and of

, intercellular junctions (black arrows). (C,D) Cytoplasmic membrane

ck arrows), and severe degenerative swelling of mitochondria (**) and

endoplasmic reticulum (black arrowheads). (E) Severe swelling of the

ack arrow) and rupture of the nuclear envelope (black open arrow). (F)

le structure. Nucleoplasm (n), nucleolus (Nc), nuclear envelope (white

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4. Discussion

Ovine embryos have been subjected to cryopre-

servation using both controlled slow freezing and OPS

vitrification [1,9,10,11]. With the present native sheep

breed, the Portuguese Black Merino, we have found no

differences in lambing (55–68%) and embryo survival

(36–45%) rates after the transfer of embryos cryopre-

served by both methods (data not shown), which is in

agreement with previous studies in other sheep breeds

[1,9,11]. The relatively high pregnancy rates obtained

after transfer of cryopreserved embryos using both

techniques, suggests that cryopreservation and warm-

ing do not seriously affect embryo viability [1].

However, in most of the studies that compared

different cryopreservation methods, embryo reselec-

tion after thawing has been performed [9,11], and in

some cases only blastocysts that had reexpanded were

transferred [10], which may result in some embryos

being wrongly discarded based on post-thawing

morphological scorings [1]. On the other hand, a

few embryos appearing as morphologically good after

thawing fail to produce pregnancies [8], which also

might be due to the transfer of embryos with serious

ultrastructural lesions not detected at the lower

resolution given by stereomicroscopy.

Although evaluation of embryo morphology in

semithin sections is not a viable method to select

embryos for transfer, it allows the identification of cell

and subcellular injuries that might not be detected by

stereomicroscopy of living embryos at thawing and so,

it can be a useful tool to improve our knowledge of the

lesions resulting from different cryopreservation

methods [8,21]. In the present study we found

dissimilar embryo grading classifications after semi-

thin evaluation of embryos previously graded by

stereomicroscopical scoring, which confirms the

usefulness of the semithin evaluations. These differ-

ences support previous reports [1,8] wherein some

embryos with a poor/fair grade at stereomicroscopical

scoring are still viable while others with a good score

are developmentally compromised [14,15]. This may

be due, at least in part, to misclassification after

stereomicroscopical scoring.

The ultrastructural analysis of cryopreserved–

thawed embryos is limited to ovine and bovine embryos

cryopreserved by controlled slow freezing [15,22],

and vitrified bovine and swine embryos [14,21,23]. In

the present study, we have described the ultrastructural

characteristics and the cryoinjuries observed in

grades 1, 2 and 3 in vivo produced ovine morulae

and blastocysts, after OPS vitrification and thawing.

Please cite this article in press as: Bettencourt EMV, et al. Ultras

produced ovine embryos. Theriogenology (2008), doi:10.1016/j.t

Independently of the stage of development considered

and cryopreservation method used, embryos scored as

grade 1 at semithin evaluation showed well preserved

blastomeres with an intact structure, while embryos

scored as grade 3 presented degenerated cells, disrupted

cell membranes, severe mitochondrial injury and

cytoskeleton disorganization, which confirm results

from previous reports using controlled slow frozen

ovine and bovine embryos [15,24], vitrified bovine

embryos [14], and swine embryos vitrified by OPS

[21,25]. At the ultrastructural level, there were no

differences between both cryopreservation methods for

embryos with the same grade after semithin scoring.

Despite the absence of studies specifically comparing

the ultrastructure of embryos cryopreserved by con-

trolled slow freezing or vitrification, similar cellular

lesions were described for bovine, ovine and porcine

embryos cryopreserved by both methods

[14,15,21,22,23]. In addition, OPS vitrified swine

embryos classified as degenerated (grade 3) following

thawing presented similar cellular lesions, namely

badly defined cellular limits, poor intracellular pre-

servation and presence of cell debris [21].

Although cryoprotectants are known to affect

cytoskeleton dynamics, the interaction between cytos-

keleton dynamics and the cellular organelles of

embryos are not consistent between species and stages

of embryo development [3,24]. Nevertheless, the early

injuries here described, such as loss of microvilli,

intercellular junctions and cytoplasmic membrane

stability, could have a disruptive impact over the

cytoskeleton, which would then lead to a severely

disturbed cell organization such as that found in

degenerated blastomeres [25]. The present data also

suggests an early loss of the mitochondrial structure,

which leads to disruption of energy supply to cell

boundaries. Damaged mitochondria after cryopreserva-

tion had been reported in previous studies [14,15,21],

suggesting that the preservation level of the mitochon-

drial matrix and envelope can be used as a reliable sign

of cellular damage. Accordingly, stages showing higher

degrees of cell damage were here associated with more

severe mitochondrial injuries that ranged from severe

matrix swelling, with loss of cristae, to membrane

degeneration and rupture. This was accompanied by a

progressive degenerative process that involved swelling

of light vesicles and endoplasmic reticulum cisternae,

followed by swelling of the nucleoplasm, loss of nuclear

pore complexes and of the nucleolar structure, and

nuclear envelope disruption.

Semithin and transmission electron microscopy

evaluation of OPS vitrified bovine and swine blastocysts

tructural characterization of fresh and cryopreserved in vivo

heriogenology.2008.10.019

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+ Models

THE-10884; No of Pages 12

were performed with embryos that were cultured in

vitro for 4 and 24 h after thawing [14,21,22]. With these

in vitro culture periods, clear signs of regeneration were

observed at the ultrastructural level, even in embryos

whose stereomicroscopy evaluation at thawing indi-

cated signs of severe damage. These signs of

regeneration included the reestablishment of junctional

contacts between trophectoderm cells and normal-

ization of the mitochondrial morphology [14,25]. The

present study was performed only after 1 h of culture

after thawing, to better simulate the clinical field

situation. It is possible that long term embryo recovery

then occurred in the uterine cavity, as it may happen in

vitro [14,25], which would help to explain the similar

rates of lambing and embryo survival after transfer of

embryos cryopreserved by both methods [1,9,11].

However, this is not necessarily always the case, as

some of the lesions found in this study, in particular for

embryos scored as grade 3, were hardly susceptible of

repair.

In conclusion, this study describes for the first time

the semithin and ultrastructural evaluation of OPS

vitrified ovine embryos, classified with different scores

at thawing (from grades 1 to 3). The utrastructural

lesions found for OPS-vitrified and controlled slow

frozen embryos were similar, independently of embryo

stage, and affected determinant cell components, such

as mitochondria and the cytoskeleton, which can

influence vital cell functioning and thus compromised

embryo viability after cryopreservation. The extension

and gravity of the cellular damage observed at the

ultrastructural level were proportional to those observed

at semithin sections performed on cryopreserved

embryos fixed after thawing. Furthermore, embryo

grading was dissimilar between stereomicroscopical

and smithin embryo scoring. Therefore, the semithin

scoring can be a useful tool to better evaluate

ultrastructural lesions in cryopreserved embryos. Most

of the cryopreserved embryos presented minor cellular

lesions. However, others displayed severe and extensive

injuries (grade 3 embryos), especially blastocysts

submitted to OPS vitrification. Therefore, cryopreser-

vation methods must be chosen according to the species

and embryonic stage and improvements in cryopreser-

vation and thawing methods to decrease cellular

damage of ovine embryos are still warranted.

Acknowledgements

This work was partially supported by funds from the

Portuguese AGRO projects (AGRO 438 Portuguese

Animal Germplasm Bank), FEDER and FCT (SFRH/

Please cite this article in press as: Bettencourt EMV, et al. Ultras

produced ovine embryos. Theriogenology (2008), doi:10.1016/j.t

BD/30575/2006; POCI/SAU-MMO/60709,/60555 and/

59997/04; UMIB).

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