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Original Paper Veterinarni Medicina, 53, 2008 (7): 358–364
358
In vitro production of embryos from high performance
cows and the development of frozen-thawed embryos
after transfer: a field study
M. M1, P. H1, Z. R1, K. H1,
J. S2, R. P3
1Veterinary Research Institute, Brno, Czech Republic
2University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic
3Palacky University, Olomouc, Czech Republic
ABSTRACT: In this field study, embryos were derived from genetically highly valuable cows excluded from breed-
ing due to reproductive disorders. Cows, 5 to 10 years old, of Czech Siemmental, Holstein Dairy and Beef Cattle
breeds were used as oocyte donors. Oocytes were obtained either in the growth phase of the first follicular wave
from cows with synchronized oestrus or in any other phases of follicular development from cows without oestrus
synchronization. The embr yos were prepared by a standard protocol described previously. The mean number of
usable oocytes, transferable and freezable embryos per donor, and the mean percentage of usable, transferable and
freezable embr yos were assessed. The results were analyzed by Student’s-t and Chi-squared tests. The embryos
were frozen according to a slow freezing protocol. After thawing, they were transferred to recipients on Day 7 after
oestrus. Irrespective of the breed, the mean numbers of usable oocytes and transferable and freezable embr yos
collected per donor were significantly higher (P < 0.01) for the synchronized than for the nonsynchronized donors
(20.4 vs 11.7, 4.3 vs 1.0 and 3.2 vs 0.8, respectively). Similarly, the mean percentages of usable oocytes, transfer-
able and freezable embryos were significantly higher (P < 0.01) for the synchronized than for the nonsynchronized
donors (28.5% vs 20.5%, 20.9% vs 9.0% and 15.8% vs 6.5%, respectively). On comparison of the synchronized and
nonsynchronized donors of each breed, the difference in the mean percentage of usable oocytes was significant
(P < 0.01) in cows of all three breeds, the difference in the mean percentage of transferable embryos was significant
in Czech Siemmental and Holstein Dairy cows (P < 0.01) and the difference in the mean percentage of freezable
embryos was significant only in Holstein Dairy cows (P < 0.01). After the transfer of 41 frozen-thawed embryos
and 43 fresh embryos, 20 heifers and 24 heifers became pregnant, respectively. In conclusion: (a) higher number
of ooc ytes from infertile, genetically valuable cows was recovered in the growth phase compared with the other
phases of follicular development; (b) greater development of these oocytes resulted in more embryos for transfer
and cryopreservation; (c) the transfer of frozen-thawed and fresh embryos resulted in pregnancy rates of 48.8%
and 55.8% , respectively.
Keywords: cattle; in vitro embryos; cryopreservation; embryo transfer
Supported by the Ministry of Agriculture of the Czech Republic (Grants No. 1B44034 and No. 02716201).
At present, reproductive biotechnologies are wide-
ly used for farm animal breeding. In cattle methods
such as artificial insemination or superovulation
and embryo transfer are generally employed, be-
cause they allow us to utilize the genetic potential
of male and female animals more effectively. The
in vitro production of embr yos from oocytes of
genetically highly valuable donors eliminated from
breeding because of reproductive disorders can ac-
celerate genetic progress. However, this requires an
increase in the efficiency of usable oocyte collec-
tion, transferable embryo production and embryo
Veterinarni Medicina, 53, 2008 (7): 358–364 Original Paper
359
survival after cryopreservation. Up to now, the pro-
duction of embryos from oocytes of selected cows
has not been very effective in comparison with the
production of embryos from oocytes of popula-
tions of ordinary slaughter cows. Furthermore, in
vitro produced embryos have lower cryotolerance
than in vivo produced embryos (Enright et al., 2000;
Dobrinsky, 2002), because of their higher sensi-
tivity to low temperatures (Leibo and Loskutoff,
1993; Holm and Callesen, 1998). Embryo survival
after cryopreservation has been shown to depend
on the quality, age and developmental stage of an
embryo at freezing (Fahning and Garcia, 1992;
Mahmoudzadeh et al., 1995; Dinnyes et al., 1999;
Ravindranatha and Reddy, 2001; Machatkova et al.,
2006). Both the embryo quality and the kinetics
of embryo development are related to the oocyte
and, most importantly, to its meiotic and devel-
opmental competence (Rizos et al., 2002; Sirard et
al., 2006; Lonergan, 2007). It has been documented
that follicles present on the ovaries at the end of
the growth phase of follicular wave can provide
oocytes with greater meiotic and developmental
competence (Hagemann et al., 1999; de Wit et al.,
2000; Machatkova et al., 2004).
The efficiency of oocyte collection as well as of
fresh and frozen embryo production were com-
pared between the oocytes recovered in the growth
phase and those recovered in the other phases of
follicular development from genetically highly
valuable cows excluded from breeding due to re-
productive disorders. The frozen-thawed viability
of these embr yos was verified by the pregnancy
rate in recipients after embryo transfer.
MATERIAL AND METHODS
Cows
A total of 52 cows, between 5 and 10 years of
age, of Czech Siemmental (n = 15), Holstein Dairy
(n = 29) and Beef Cattle (n = 8) breeds, were used
as oocyte donors. In the first group of donors
(n = 31), the oestrous cycle was synchronized by
two doses of PGF2α at an interval of 11 days and
one dose of hCG (Pregnyl, 1 500 IU, Organon, Oss,
The Netherlands). The oocytes were collected on
Days 3–4 after oestrus in the growth phase of the
first follicular wave. Subsequently, the cows were
slaughtered. Evaluation criteria for the growth
phase were the presence of a hemorrhagic corpus
luteum with signs of ovulation and no dominant
follicle on the ovaries larger than 11 mm in diam-
eter. In the second group of donors (n = 21), the
oestrus was not synchronized before slaughter and
oocytes were collected in any of the other phases
of follicular development.
Bulls
The frozen-thawed sperm of 21 elite bulls of
Czech Siemmental (n = 6), Holstein Dairy (n = 8)
and Beef Cattle (n = 7) breeds were used for oocyte
fertilization.
Embryo production
Oocyte-cumulus complexes were isolated by to-
tal slicing of the ovarian cortex. Only good quality
oocytes with homogenous dark cytoplasm and at
least two layers of cumulus cells were considered us-
able. Embryos were prepared according to a stand-
ard protocol described previously (Machatkova et
al., 2004). Oocytes were matured in TCM-199 me-
dium (Earle’s salt), supplemented with antibiotics,
0.20mM sodium pyruvate (Sigma-Aldrich, St. Louis,
MO, USA), gonadotropins (P.G. 600, 15 IU/ml,
Intervet, Boxmeer, The Netherlands) and 5% oe-
strus cow serum (ECS) for 24 hours.
Motile spermatozoa were isolated by the swim-up
method from frozen-thawed sperm using modified
Tyrode’s medium (SP-TALP). They were coincu-
bated with the oocytes at a concentration of 1 × 106
spermatozoa per ml in modified Tyrode’s medium
(IVF-TALP) supplemented with 10 g/ml heparin
for 20 hours. Presumptive zygotes were removed
from cumulus cells, transferred onto a BRL cell line
monolayer (Buffalo rat liver cells, ATCC, Rockville,
MD, USA) and cultivated in Menezo B2 medium
with 10% ECS. All procedures were carried out
at 39°C with atmospheric conditions of 5% CO2.
Embryos, at earliest at an early blastocyst stage on
Day 7, or advanced blastocyst stage on Day 8 were
regarded as transferable.
Embryo cryopreservation
Only excellent blastocysts of good quality were
selected for cryopreservation from transferable
embryos. They were placed in freezing medium,
Original Paper Veterinarni Medicina, 53, 2008 (7): 358–364
360
consisting of 10% glycerol (v/v) in TCM-199 me-
dium with 10% ECS and equilibrated for 5 min at
room temperature. Subsequently, each embryo was
loaded into 0.25 ml straw in a column of freezing
medium and allowed to stand for another 10–15
min at room temperature. Straws were placed in the
programmable freezer at –7°C and, after 10 min,
were seeded. After another 10 min the embryos
were cooled to –35°C at a rate of 0.3°C/min; they
were then plunged into liquid nitrogen.
Embryo transfer
The embryos were thawed by holding the straws
for 10 s in the air and then placing them in a 30°C
water bath for 30 s. The cryoprotective was re-
moved using a three-step procedure (6.6% glycerol
[v/v] and 0.3M sucrose; 3.3% glycerol and 0.3M su-
crose; 0.25M sucrose in culture medium, TCM-199
with 10% ECS). Subsequently, the embryos were
washed with culture medium and transferred as
soon as possible to heifers on Day 7 after their oe-
strus. The pregnancy rate was assessed by palpation
per rectum on Day 90 after transfer.
Statistical analysis
The mean number of usable oocytes , transfer-
able and freezable embryos per donor, and the
mean percentage of usable oocytes from collected
oocytes, transferable and freezable embryos from
usable oocytes were assessed. The results were ana-
lyzed by Student’s-t and Chi-squared tests, using
ANOVA SPSS Version 11.5 software for Windows
(SPSS, Inc., Chicago, IL, USA).
RESULTS
Oocytes
The efficiency of oo cyte collection from syn-
chronized and nonsynchronized donors of dif-
ferent breeds is shown in Table 1. Regardless of
the breed, the mean numb er of usable ooc ytes
collected per donor and the mean percentage of
usable oocytes were significantly higher (P < 0.01)
for the synchronized than for the nonsynchronized
donors. An evaluation of each breed revealed that
the mean number of usable ooc ytes per donor
was higher for the synchronized donors of Czech
Siemmental, Holstein Dairy and Beef Cattle breeds,
but that only in Czech Siemmental cows was the
increase significant (P < 0.05). The difference in
the mean percentage of usable oocytes between
the synchronized and nonsynchronized donors was
significant in cows of all breeds (P < 0.01, P < 0.05
and P < 0.05, respectively).
Table 1. Collection of oocytes from donors of different breeds in the growth phase and other phases of follicular
development
Breed
Synchronized donors Nonsynchronized donors
donors
(n)
oocytes
donors
(n)
oocytes
isolated usable usable/iso-
lated
(mean %)
isolated usable usable/iso-
lated
(mean %)
total
(n)
per donor
(n)
total
(n)
per donor
(n)
total
(n)
per donor
(n)
total
(n)
per donor
(n)
Czech
Siemmental 9 592 65.8 182 20.2a1 30.7a1 6 366 61.0 64 10.7b1 17.5c1
Holstein
Dairy 17 1 380 81.2 352 20.7a1 25.5a2 12 738 61.5 156 13.0a1 21.1b1
Beef Cattle 5 245 49.0 98 19.6a1 40.0a3 3 93 31.0 25 8.3a1 26.9b 2
Total 31 2 217 71.5 632 20.4a28.5a21 1 197 57.0 245 11.7c20.5c
Values in the same row with different superscripts are significantly different (a–bP < 0.05; a–cP < 0.01)
Values in the same column with different superscripts are significantly different (1–2P < 0.05; 1–3, 2–3P < 0.01)
Veterinarni Medicina, 53, 2008 (7): 358–364 Original Paper
361
Transferable embryos
The efficiency of transferable embryo production
from the synchronized and nonsynchronized do-
nors of evaluated breeds is summarized in Table 2.
Regardless of the breed, the mean number of trans-
ferable embryos produced per donor and the mean
percentage of transferable embryos were signifi-
cantly higher (P < 0.01) for the synchronized than
for the nonsynchronized donors. The mean number
of transferable embryos per donor was higher for
the synchronized donors of all three breeds, but
only in Czech Siemmental cows was the value
significant (P < 0.05). The difference in the mean
percentage of transferable embryos was significant
in cows of Czech Siemmental and Holstein Dairy
breeds (P < 0.01).
Freezable embryos
The efficiency of freezable embryo production
from the synchronized and nonsynchronized do-
nors of different breeds is presented in Table 3.
Regardless of the breed, the mean number of freez-
able embryos produced per donor and the mean
percentage of freezable embryos were significantly
higher (P < 0.01) in the synchronized than the non-
Table 2. Collection of transferable embryos from donors of different breeds in the growth phase and other phases
of follicular development
Breed
Synchronized donors Nonsynchronized donors
donors
(n)
oocytes
(n)
embryos
donors
(n)
oocytes
(n)
embryos
transferable transferable/
oocytes
(mean %)
transferable transferable/
oocytes
(mean %)
total
(n)
per donor
(n)
total
(n)
per donor
(n)
Czech
Siemmental 9 182 43 4.8a1 23.6a 1 6 64 8 1.3b1 12.5c1
Holstein
Dairy 17 352 76 4.5a1 21.6a1 12 156 13 1.1a1 8.3c1
Beef Cattle 5 98 13 2.6a 1 13.3a2 3 25 1 0.3a1 4.0a1
Total 31 632 132 4.3a20.9a21 245 22 1.0c9.0c
Values in the same row with different superscripts are signicantly different (a-bP < 0.05; a-cP < 0.01)
Values in the same column with different superscripts are significantly different (1-2P < 0.05)
Table 3. Collection of freezable embryos from donors of different breeds in the growth phase and other phases of
follicular development
Breed
Synchronized donors Nonsynchronized donors
donors
(n)
oocytes
(n)
embryos
donors
(n)
oocytes
(n)
embryos
freezable freezable/oo-
cytes
mean %
freezable freezable/
oocytes
(mean %)
total
(n)
per donor
(n)
total
(n)
per donor
(n)
Czech
Siemmental 9 182 31 3.4a1 17.0a1 6 64 5 0.8a1 7.8a1
Holstein
Dairy 17 352 59 3.5a1 16.8a1 12 156 10 0.8a1 6.4c 1
Beef Cattle 5 98 10 2.0a1 10.2a1 3 25 1 0.3a1 4.0a1
Total 31 632 100 3.2a15.8a21 245 16 0.8c6.5c
Values in the same row with different superscripts are signicantly different (a-cP < 0.01)
Values in the same column with the same superscripts are not significantly different
Original Paper Veterinarni Medicina, 53, 2008 (7): 358–364
362
synchronized donors. A separate evaluation of each
breed showed that the mean number of freezable
embryos per donor was higher for the synchronized
donors of all three breeds, but that the difference
was not significant. The difference in the mean
percentage of freezable embryos was significant
in Holstein Dairy cows (P < 0.01).
Embryo development after transfer
The frozen-thawed viability of the embryos was
determined by their development after transfer
into heifers on Day 7 after their oestrus. From the
synchronized donors, 41 frozen-thawed embryos
were transferred and 20 recipients became preg-
nant (48.8%). The transfer of 43 fresh embryos de-
rived from the synchronized donors resulting in
24 pregnancies (55.8%) served as a control.
DISCUSSION
The application of new biotechnologies in cattle
breeding can intensify the selective process and
bring about a genetic benefit. The in vitro produc-
tion of embryos from high performance donors,
which cannot meet reproductive condition and
must, therefore, be eliminated from breeding and
slaughtered, is one of these methods. Combining
this method with cryopreservation enhances the
utilization of embryos from genetically valuable
parents for both the preser vation of genetic re-
sources for the future and the utilization of embryo
transfers within ongoing breeding programs. Up to
now, however, the development of embryos pro-
duced in vitro from oocytes of selected donors and
survival of these embryos after cryopreservation
have been low compared with embryos produced
in vivo from superovulated donors (Hasler, 2001;
Dobrinsky, 2002).
In order to increase the developmental ability
of in vitro produced and cryopreserved embr yos,
selection based on the kinetics of embryo devel-
opment or the modification of conditions during
maturation, cultivation and cryopreservation are
recommended (Massip et al., 1995; Rizos et al., 2001,
2003; Cho et al., 2002; Imai et al., 2002; Mtango et
al., 2003; Nedambale et al., 2004; Tominaga et al.,
2007). New strategies based on metabolic manipu-
lation with embryos before their cryopreservation
have been described by Seidel (2006).
In our experiment s, we made an effort to in-
crease the production of embryos and to improve
the survival of these embryos after freezing by the
selection of oocytes with greater meiotic and de-
velopmental competence.
In our preliminary study involving slaughtered
cows we found that oocytes recovered in the growth
phase of the first follicular wave and fertilized by
the spermatozoa of a standard bull produced more
embryos with accelerated development and bet-
ter morphological quality than oocytes recovered
in the other stages of follicular development. We
concluded that this approach could be used as an
alternative method for the production of cryop-
reser ved embryos from high performance cows
(Machatkova et al., 2006).
In our field study with reproductively problem-
atic donors, the 52 unfertile, genetically valuable
cows provided a total of 877 usable oocytes, giv-
ing rise to 154 fresh embryos, 116 of which were
used for cryopreservation (17.6% and 13.2%, re-
spectively). Although the number of usable oocytes
collected per donor was similar in all three breeds,
the numbers of transferable and freezable embryos
per donor obtained from Czech Siemmental and
Holstein Dairy cows were twice the numbers of
those obtained from Beef Cattle cows; this is be-
cause embryo development rates were significantly
higher for the first two breeds than for the third
one. For Czech Siemmental, Holstein Dairy and
Beef Cattle cows, the mean proportion of transfer-
able embryos derived from oocytes recovered in
the growth phase of follicular development were
23.6%, 21.6% and 13.3%, respectively. These val-
ues are comparable with those reported by Galli
et al. (2003) who obtained embryo development
rates of 21.8% and 15.6% in oocytes collected from
culled genetically valuable cows and heifers of one
herd, respectively, and by Merton et al. (2007) who
reached embryo development rates of 23.4 to 24.7%
in oocytes collected by transvaginal aspiration from
healthy pregnant heifers and first-parity Holstein
Friesian cows.
It is generally known that the pregnancy rate af-
ter the transfer of cryopreserved embryos is lower
than the rate after transfer of fresh embryos and
that it is significantly lower after the transfer of
frozen in vitro produced compared to the transfer
of in vivo produced embryos (Hasler, 2001; Riha
et al., 2002; Galli et al., 2003; Merton et al., 2007).
Pregnancy rates of 28%, 30–40%, 32.8–39.2% and
35.6–40.8% were achieved by Dobrinsky (2002),
Veterinarni Medicina, 53, 2008 (7): 358–364 Original Paper
363
Hernandez-Fonseca et al. (2002), Hoshi (2003) and
Merton et al. (2007), respectively, after the transfer
of frozen-thawed embryos produced in vitro from
slaughterhouse-collected and transvaginally aspi-
rated oocytes.
In this study the pregnancy rates were 48.8% for
cryopreserved and 55.8% for fresh embryos. A rela-
tively low decrease in viability was found for frozen-
thawed embryos compared to fresh embryos. The
pregnancy rate was improved after the transfer of
frozen-thawed embryos derived from oocytes with
greater meiotic and developmental competence in
comparison with the pregnancy rates after transfer
of embryos derived from oocytes with heteroge-
neous meiotic and developmental competence, as
used for embryo production by the authors men-
tioned above.
The results of this study confirm our assumption
that the recovery of oocytes in a suitable phase of
follicular development can be used to increase the
production of fresh and frozen embryos from high
performance donors and to improve the pregnancy
rate after embryo transfer. In this case, however,
the examination of follicular status of selected cows
and the timing of oocyte collection for embryo
preparation are important.
It can be concluded that (a) higher numbers of
usable oocytes from infertile, genetically valuable
cows can be recovered in the growth phase com-
pared with any other phase of follicular develop-
ment; (b) greater de velopment of these ooc ytes
results in more embryos for transfer and cryop-
reservation; (c) the pregnancy rate can be improved
after the transfer of frozen-thawed embryos derived
from oocytes with greater meiotic and develop-
mental competence.
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Corresponding Author:
Ing. Marie Machatkova, CSc., Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic
Tel. +420 533 331 418, fax +420 541 211 229, e-mail: machatkova@vri.cz
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Received: 2008–04–18
Accepted after corrections: 2008–06–11