Improved vitrification method allowing direct transfer
F. Guignot , A. Bouttier , G. Baril P. Salvetti P. Pignon
J.F. Beckers , J.L. Touze´ , J. Cognie´ , A.S. Traldi
a INRA-CNRS-Universite´ de Tours-Haras Nationaux, Physiologie de la Reproduction et des Comportements, 37380 Nouzilly, France
b Universite´ de Lie`ge, Faculte´ de Me´decine Ve´te´rinaire, Physiologie de la Reproduction, B-4000 Lie`ge, Belgium
c Dep. de Reproduc¸ao Animal, FMVZ-USP, cp 23, 13630 000 Pirassununga, SP, Brazil
Received 30 September 2005; received in revised form 24 February 2006; accepted 26 February 2006
The aim of this study was to design a vitrification method suited to field embryo transfer experiments in goat. In a first
experiment, a standard vitrification protocol, previously designed for sheep embryos was compared to slow freezing of goatembryos. No significant difference was observed on kidding rate (48% versus 69%, respectively), nor on embryo survival rate (35%versus 45%). Second experiment: all embryos were vitrified. After warming, embryos were either transferred directly (directtransfer), or after in vitro dilution of the cryoprotectants (conventional transfer). The kidding rate was not affected by the transfermethod (38% versus 23%, respectively). However, embryo survival rate tended to be higher after direct transfer (26% versus 14%).
Third experiment: OPS vitrification was compared to standard vitrification. The kidding rate was not affected (22% versus 39%,respectively), but the embryo survival rate was lower after OPS (14% versus 28%). Fourth experiment: 0.4 M sucrose was addedwith cryoprotectants in vitrification. The kidding rate after direct transfer was significantly enhanced after addition of sucrose (56%versus 27%, respectively), whereas embryo survival rate was not significantly affected (32% versus 18%). Fifth experiment:vitrification with sucrose supplementation was compared to slow freezing. No significant difference was observed after directtransfer on kidding rate (52% versus 31%, respectively), but embryo survival rate tended to be higher after vitrification (34% versus21%). In conclusion, our results indicate that addition of 0.4 M sucrose in association with direct transfer improves significantly theviability of goat vitrified embryos.
# 2006 Elsevier Inc. All rights reserved.
Keywords: In vivo goat embryo; Vitrification; Slow freezing; Transfer; Embryo survival
settings. Embryo cryopreservation has simplified themanagement of genetic resources in domestic and wild
Cryopreservation and thawing under controlled
species and is essential in commercial embryo transfer
conditions have permitted the use of cells and tissues
technology providing easier and cheaper embryo
after long-term storage in both commercial and research
transport, reducing health risk and avoiding loss ofanimals during transport. In domestic animals itprovides methodology to maintain genetic diversityconservation and preservation of endangered species it
* Corresponding author. Tel.: +33 2 47 42 77 94;
enables maintenance and storage of biodiversity via
0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved.
F. Guignot et al. / Theriogenology 66 (2006) 1004–1011
Cryopreservation of mammalian embryos was
Many different vitrification protocols have been used
developed during the 1970s in mouse and cow
with success to cryopreserve in vivo and in vitro
and has since been modified to attempt to cryopreserve
produced embryos in several species. All of these
embryos from most species, at different developmental
protocols aim to minimize the toxicity due to high
stages, as well as more sensitive embryos such as in
cryoprotectant concentrations. A lower temperature of
vitro produced, cloned or biopsied embryos
vitrification, an accelerated rate of cooling, a combina-
tion of several cryoprotectants rather than a single one,
slow freezing which is designed to maintain a delicate
partial replacement of cryoprotectants by macromole-
balance between cryoprotectants at low concentration
cules (PVP, Ficoll) and sugars to increase the viscosity
(1–1.5 M) and aqueous embryo compartment, whereas
of the vitrification medium have been proposed to
the strategy of the vitrification method is a rapid
solidification of liquid with high concentrations of
cryoprotectants (6–7.5 M) avoiding ice crystal forma-
vitrification solution seems to be the most critical
tion. In goat, the first successful cryopreservation of
factor to determine the success of vitrification. Some
embryos was with the slow freezing method in 1976 .
studies have specially noticed the positive effect of
Subsequently, most goat embryos were cryopreserved
sugar addition in vitrification solution, as trehalose or
by slow freezing until 1990 with a rate of embryo
0.1 and 0.3 M sucrose in the last step of equilibration
1990, the first successful transfer of vitrified goat
procedure has significantly enhanced the hatching rate
embryos was reported by Yuswiati and Holtz . They
of Day 6 and Day 7 embryos as compared to 0.0 and
used standard vitrification with ethylene glycol (EG)
0.5 M sucrose . To our knowledge, vitrification with
and propanediol, in 0.25 mL straw. In 2001, the first
addition of sugar in the last step of equilibration
successful transfer of goat embryos using open pulled
procedure has never been tested with goat embryos.
straw (OPS) technique was obtained by El-Gayar and
In the present study, we compared in vivo goat
Holtz . However, only limited results are available
embryo survival rate after slow freezing and vitrifica-
after transfer of goat vitrified embryos with generally
tion. We tested the effect of cryoprotectant removal
lower survival rates than those obtained after slow
before transfer compared with direct transfer and we
tried to improve the vitrification method by the addition
In small ruminants, the use of embryo transfer
of sucrose at the last step of equilibration procedure and
technique in breeding schemes is limited as compared
by using the open pulled straw (OPS) technique.
with cattle This is probably due to the excessivecost when compared to the value of the animal The
more satisfactory and commonly used technique ofcryopreservation in goats (slow freezing) is unfortu-
nately the more expensive since it requires an expensivebiological freezer and is labour intensive, whereas
The estrus cycles of Alpine and Saanen dairy goats
vitrification techniques with direct transfer offers a real
were synchronized in donor and recipient females by
possibility to reduce the cost of embryo transfer .
insertion of intravaginal sponges containing 45 mg FGA
Efficient and economical cryopreservation techniques
(Chronogest1, Intervet, Angers, France) for 12 days (day
for goat embryos would be advantageous since goats
0 = sponge insertion), and 50 mg cloprostenol (Estru-
produce diversified products of commercial interest. In
mate1, Shering-Plough, Levallois, France) were admi-
addition, emerging biotechnologies such as transgenesis
nistrated in the morning of the 10th day. Two days before
and somatic cell nuclear transfer allows for generation
sponge removal, the recipients received 400–500 IU of
and propagation of transgenic founder animals that
eCG (Chronogest1, lntervet, Angers, France). Donors
produce valuable recombinant proteins of pharmaceutic
were superovulated with a total of 160 mg pFSH (Me´rial,
or biomedical interest in goat milk Moreover,
Belgium) injected intramuscularly every 12 h in six
his short gestation period (5 months) small size, easy
decreasing doses (40, 40, 20, 20, 20, and 20 mg) during
breeding and relatively high milk production make goat
the last three days of progestagen treatment (days 10–12).
competitive as compared to the cow for the application
pFSH was supplemented by 66 mg LH in the last two
of biotechnologies. Therefore, improvement of embryo-
injections. When the onset of estrus was detected (24–
based techniques is warranted in this species, especially
36 h after sponge removal), donors were mated with
several bucks in experiments 2, 3, 4 and 5, while in
F. Guignot et al. / Theriogenology 66 (2006) 1004–1011
experiment 1, donors were inseminated in utero under
or to the two transfer methods applied in each
endoscopic control with frozen/thawed semen from
experiment. After embryo collection, local antibiotic
several fertile bucks (200 Â 106 spermatozoa per female)
(Aluspray) was spreaded on the abdominal incision, and
20–24 h after the onset of estrus.
each recipient received benzylpenicilline and dihydro-streptomycine during 3 days (i.m. 106 UI per day).
2.3. Cryopreservation and warming procedures
Embryos were recovered surgically by laparotomy
under general anesthesia (iv thiopental 10 mg kgÀ1,
The different cryopreservation and transfer methods
atropine sulphate 0.3 mg kgÀ1 and endotracheal intuba-
applied in each experiment are summarized in .
tion with isoflurane and oxygen) 7–8 days after the onset
Five to six embryos were cryopreserved together in
of estrus. Each uterine horn was flushed with 40 mL of
a same straw for conventional transfer (experiments 1
pre-warmed PBS containing 2% BSA (Euroflush, IMV,
and 2) while only two embryos were loaded in each
L’Aigle, France). The embryos were immediately
straw for direct and pseudo-direct transfer (experiments
retrieved and placed in PBS containing 4% BSA
(Embryo Holding medium, IMV, L’Aigle, France). Theywere classified according to their stage of development
2.3.1. Slow freezing (experiments 1 and 5)
and only morphologically normal embryos were selected
Embryos were frozen according to the method of Le
for cryopreservation (compact morulae, blastocysts and
Gal et al. Briefly, embryos were equilibrated in
expanded blastocysts). Embryos were kept for less than
freezing solution consisting successively of 0.5, 1 and
30 min in Embryo Holding medium at +20 to +25 8C
1.5 M EG in Embryo Holding medium, 5 min each, at
before cryopreservation. In the five experiments, the
room temperature. During the last equilibration step,
embryos from each donor were divided between two
embryos were loaded into the centre of a 0.25 mL
groups according to the two cryopreservation techniques
plastic straw (IVM, L’Aigle, France) within 20–30 mL
Table 1Pregnancy rate of in vivo produced goat embryos transferred after different protocols of cryopreservation
The letter a and b denotes the values differ significantly in each experiment (chi-square test): (P < 0.05). The symbol a and b denotes the values tendto be significant in each experiment (chi-square test): (P < 0.10). Standard vitrify.: standard vitrification without sucrose; OPS: open pulled straw.
b Ultrasound examination and PAG assay (pregnancy-associated glycoprotein).
d Defined as the kids born/thawed embryos ratio.
e Two pregnant goats were died few days before kidding.
F. Guignot et al. / Theriogenology 66 (2006) 1004–1011
of medium. Embryos were separated from two surround-
were then equilibrated in a freezing solution consisting of
ing segments of freezing medium by two air bubbles
7.5% EG and 7.5% DMSO dissolved in Hepes-buffered
(experiment 1) or of Embryo Holding medium (experi-
TCM199–NBCS for 3 min. They were then transferred
ment 5) of about 90 mL each. The straws were sealed and
with approximately 1 or 2 mL of solution into a 20 mL
placed in a programmable freezer (Agrogen-EF1,
droplet of 18% EG, 18% DMSO and 0.4 M sucrose in
Freiburg, Switzerland) and cooled from room tempera-
Hepes-buffered TCM199–NBCS. Embryos were drawn
ture (22 8C) to À7 8C at 4 8C/min. After 5 min, each
up together in another 1–2 mL drop, loaded by capillarity
straw was manually seeded. Following a further 10 min at
into the narrow end of an OPS and plunged into liquid
À7 8C, straws were cooled to À30 8C at a rate of 0.3 8C/
nitrogen. The time between the contact of the embryos
min, and finally plunged and stored into liquid nitrogen.
with the concentrated cryoprotectant solution and the
For thawing, straws were held in air for 5 s and then
liquid nitrogen did not exceed 45 s. For warming, straws
dipped into a 37 8C water bath for at least 15 s. In
were held in air for 5 s before the narrow end was
experiment 1, the content of each straw was emptied into
immerged in Hepes-buffered TCM199–NBCS with
a Petri dish containing 0.25 M sucrose in Embryo
0.2 M sucrose. After 1 min, embryos were transferred
Holding medium. After 10 min (two baths of 5 min), the
for 5 min in new Hepes-buffered TCM199–NBCS with
embryos were washed twice in Embryo Holding medium
0.2 M sucrose, following by 5 min with 0.1 M sucrose.
without sucrose for 5 min each to eliminate cryoprotec-
All warmed embryos were transferred.
tants before conventional transfer. In experiment 5,embryos were directly transferred after thawing.
2.3.4. Sucrose addition in standard vitrification(experiments 4 and 5)
2.3.2. Standard vitrification (experiments 1, 2, 3
The first and the second steps of equilibration
procedure were the same as described in the standard
The vitrification method described by Mermillod
vitrification. The third step consisted of 25% glycerol
et al. was applied. Embryos were kept at room
and 25% EG for 30 s in PBS–NBCS supplemented with
temperature for 5 min in PBS supplemented with 20%
0.4 M sucrose. Freezing and warming are identical to
new-born calf serum (NBCS). They were then vitrified
standard vitrification protocol. After warming, embryos
in three steps at room temperature as follows: 10%
glycerol for 5 min, 10% glycerol and 20% EG for 5 min,and finally 25% glycerol and 25% EG for 30 s in PBS–
NBCS. During the last step, embryos were quicklyaspirated into the centre of a 0.25 mL plastic straw
In all experiments, embryos were transferred into
(IVM, L’Aigle, France) within 20–30 mL of vitrification
synchronized Alpine and Saanen goats, 7 days after the
solution. Embryos were separated by two air bubbles
onset of estrus, in different locations (replicates). Three
from two surrounding segments of PBS–NBCS contain-
replicates were done in all experiments, except in
ing 0.8 M galactose (about 90 mL each). The straws
experiment 2 (only two replicates). The recipients were
were sealed and immediately plunged directly into
sedated with 0.3 mg kgÀ1 iv acepromazine (Calmivet1,
liquid nitrogen. For warming, straws were held 5 s in air
Vetoquinol, Lure, France). After endoscopic control of
followed by 15 s in a 22 8C water bath. For conventional
the presence of at least one functional corpus luteum,
transfer in experiments 1 and 2 (half of straws), the
embryos were transferred as follows.
straw content was expelled and embryos were incubatedduring 5 min in the content of the straw and washed
twice for 5 min in 3 mL PBS to eliminate cryoprotec-
tants before conventional transfer. In experiments 2 (the
After warming and cryoprotectant removal, the
other half of straws), 3 and 4, embryos were directly
embryo quality was evaluated under stereomicroscope
according to morphological criteria, and all degeneratedembryos, i.e. those with abnormal morphology and
disrupted membrane, were eliminated. For each
The OPS vitrification method was applied according
recipient, one to three embryos were aspirated with
to the method described by Vajta et al. . All
20 mL PBS in a glass capillary connected to a 1 mL
equilibrations were performed at 34–35 8C. Briefly,
syringe and were introduced surgically into the top of
embryos were washed twice in Hepes-buffered TCM199
the uterine horn ipsilateral to the ovary bearing at least
solution supplemented with 20% NBCS for 5 min. They
one functional corpus luteum. As five to six embryos
F. Guignot et al. / Theriogenology 66 (2006) 1004–1011
were cryopreserved together in a same straw for
were obtained with slow freezing and standard vitrifica-
conventional transfer, some good embryos were
tion, respectively. In the second experiment, the kidding
exceptionally not transferred in experiment 1 because
rate was not affected by the transfer method used
(P > 0.05): 24% versus 38% of kidding were obtainedafter conventional or direct transfer of vitrified/warmed
2.4.2. Direct transfer (experiments 2, 3, 4 and 5)
embryos, respectively. However, the rate of embryo
After warming, the whole content of the straw
survival tended to be higher after direct transfer as
(250 mL, two embryos) was deposited surgically, with a
compared to conventional transfer (26% versus 14%,
prototype instrument (IMV, L’Aigle, France), into the
respectively). The OPS vitrification method (experiment
uterine horn ipsilateral to the ovary bearing at least one
3) did not enhance the kidding rate, whereas the embryo
functional corpus luteum without embryo selection or
survival rate was significantly lower compared with
standard vitrification (14% versus 28% for OPS andstandard vitrification, respectively). The addition of
2.4.3. Pseudo-direct transfer (experiment 3: OPS
sucrose to the vitrification medium (experiment 4)
significantly enhanced the kidding rate after direct
After warming and partial cryoprotectant removal,
transfer (56% versus 27% without sucrose, P < 0.05),
all warmed embryos were transferred. For each
whereas embryo survival rate was not significantly
recipient, two embryos were aspirated with 20 mL
affected (32% versus 18.2%). Only in one replicate true
Hepes-buffered TCM199–NBCS with 0.1 M sucrose in
statistically significant difference was detected: 41%
a glass capillary the same way as conventional transfer.
(n = 22) versus 10% (n = 20) with and without sucrose,
Embryos were then introduced surgically into the top of
respectively (P < 0.05). When vitrification with addition
of 0.4 M sucrose and slow freezing were compared usingdirect transfer (experiment 5), no significant difference
was observed on kidding rate (52% versus 31%,respectively), but embryo survival rate tended to be
Pregnancy was diagnosed at day 21 after estrus by
higher after vitrification (34% versus 21%, respectively).
progesterone assay (RIA method) a female wasconsidered pregnant if progesterone concentration was
higher than 1.5 ng/mL. It was confirmed by ultrasoundexaminations on day 43 and PAG assay (pregnancy-
In the present study, embryo survival and kidding
associated glycoprotein; RIA method) on day 43
was studied to compare slow freezing and vitrification,
and 70, and finally by observation of kidding at term.
with or without addition of 0.4 M sucrose in the last step
The kidding rate was defined as the pregnant/transferred
of equilibration procedure, and OPS applied to in vivo
recipient ratio and the embryo survival rate was defined
produced goat embryos followed by either direct
as the number of kids born/thawed embryos ratio.
transfer into recipients, without cryoprotectant removal,or after conventional transfer after in vitro cryoprotec-
tants removal and embryo quality evaluation. Embryosurvival rates obtained in the first experiment after slow
This study was designed to compare two cryopre-
freezing and conventional transfer (45%) were compar-
servation protocols in each experiment. Comparisons
able to results reported by Baril et al. (47.3%) with
among protocols were performed using the Chi-square
exactly the same freezing/thawing/transfer method used
test. Statistical significance was denoted as P < 0.05.
in our study, and by Holm et al. (53%) for Angoragoat and McKelvey and Bhattacharyya (53%) for
Cashmere goat after slow freezing with ethylene glycol.
Similarly, embryo survival rate after vitrification
The rate of pregnancy obtained in each experiment is
observed here (35%) was also comparable to the results
showed in . In experiment 1, 83% and 88% of
reported previously by Traldi (40%) and Branca
thawed embryos were transferable after slow freezing
et al. (44%) with the same method. In contrast, the
and standard vitrification, respectively. After conven-
embryo survival rate obtained in our second experiment
tional transfer, there was no significant difference
(14%), using the same standard vitrification technique
between cryopreservation techniques: 69% versus 48%
and conventional transfer, was markedly lower than
of kidding and 45% versus 35% of embryo survival rates
those reported by Traldi and those obtained in our
F. Guignot et al. / Theriogenology 66 (2006) 1004–1011
first experiment using slow freezing. Human error was
sucrose acts in the same way as galactose contained in the
ruled out because this experiment was conducted by the
marginal parts of the straw during vitrification procedure,
same technical team, suggesting that the difference
due to its osmotic properties. These non-permeable
observed may be related to the different batches and
sugars prevent excess water inflow because water
breeds of recipient females. Indeed, recipients were not
permeates more rapidly than cryoprotectant diffuses
the same between these two experiments and moreover
out of the embryo, reducing membrane damage and
they did not come from the same farm. Differences in
increasing the embryo survival Another advantage
embryo quality (ability to survive vitrification) could
of sucrose, and other carbohydrates, is the preservation of
also contribute to the variability observed between
the structural and functional integrity of membranes at
experiments. In addition, age of donors and season of
embryo production may have affected embryo quality.
The low embryo survival rate obtained after slow
Although the same person vitrified all embryos, the
freezing in experiment 5 (21%), compared to that
concentration of cryoprotectants was very high in this
obtained in the first experiment (45%) and reported in
method (7.5 M) and in the last step of equilibration
literature (27–59%) is difficult to explain. The
procedure the additional seconds that were required
variability of the in vivo produced embryos quality and
could induce (1) excessive dehydratation of the embryo,
the different recipient batches could perhaps explain a
resulting in an increased salt concentration into embryo
part of this unexpected difference as suggested above.
cells, which may be detrimental to protein integrity
However, as the embryos from each donor were equally
, and (2) an increase of intracellular cryoprotectants
distributed between both cryopreservation methods, it
concentration which are known to damage the embryos,
could be envisioned that embryo survival after 0.4 M
even if the chosen cryoprotectants, ethylene glycol and
sucrose vitrification, which had a tendency to be higher
glycerol, are the least toxic compared to DMSO or
than after slow freezing, could be even better with
propylene glycol . The OPS technique applied in
experiment 3 did not affect the rate of embryo survival
The pregnancy rate decreased dramatically by about
compared to standard vitrification (14% versus 28%).
20% between days 21 and 43 in experiments 1 and 4
This result differed from those obtained by El Gayar and
(standard vitrification) and in experiment 3 (OPS). As
pregnancy rate was diagnosed by progesterone assay on
elevated rates. In our experimental conditions, OPS
day 21, this variation could be attributed to a longer
technique was not suited for goat embryo cryopreserva-
luteal phase of the recipients, responsible of high level
tion. To avoid these problems of variability, we have
of progesterone at day 21, whereas embryos were no
attempted to improve the vitrification technique in
more viable. It could be also attributed to a pseudo-
experiments 4 and 5. In these experiments, a repeatable
gestation, a phenomenon which occurs often in goats
embryo survival rate of 32% and 34% was obtained
as a result of a growing trophoblastic vesicle
after addition of 0.4 M sucrose in the last step of
without a viable embryo. Because of this complication,
equilibration procedure and direct transfer to a total of
the most accurate test of early pregnancy was
54 recipient goats. Moreover, the addition of 0.4 M
ultrasound examination or PAG on day 40–43.
sucrose in the last step increased the kidding rate as
Despite the low overall survival rates obtained in
compared to standard vitrification (without sucrose) and
experiment 2, direct transfer of the embryos seemed to
tended to enhance embryo survival rate as compared to
be as effective as conventional transfer after cryopro-
tectant removal. This result was previously observed in
As shown by Martinez et al. in bovine embryos,
goats after slow freezing and in sheep after
sucrose confers an additional protective effect for the
standard vitrification In cattle, several freezing
embryos during vitrification. Addition of sucrose may
methods have been designed to allow direct transfer of
reduce the toxicity of the high level of cryoprotectants
the embryos , even under field conditions
applied in this technique, probably by reducing the
As no embryo selection is done after thawing,
intracellular amount of cryoprotectant . Indeed, the
direct transfer can be performed without specific
extra osmolarity created by the non-permeable carbohy-
expertise in embryo quality evaluation, reducing the
drate causes embryo dehydratation and rapid shrinkage
cost of the transfer, avoiding embryo elimination after
and thereby reduces the amount of cryoprotectants in the
thawing and simplifying the embryo transfer method in
cells Embryo dehydratation caused by introduc-
field conditions. Moreover, in sheep, this technique
tion of this non-permeable carbohydrate also reduces the
allows a potential gain of 7–8% in terms of lambs born
risk of ice crystal formation . During warming,
which could increase the gain/cost ratio .
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