Table 1 Comparison of in vitro development of Day 3 IVF-derived bovine embryos co-cultured with or without MEF
for 5 days Group
Cell stage No. of embryos
No. of embryos Developmental
a
Ž . cultured
developed to rate
b b
b b
EB MB
EX HB
d,e
CR1aa 4-cell
91 1
1 2
4.4
d
8-cell 103
1 2
15 6
23.3
f
Sub-total 194
1 3
16 8
14.4
e
MEF 4-cell
96 1
1 2.1
c
8-cell 105
1 25
21 44.8
g
Sub-total 201
1 26
22 24.4
a
No. of total blastocystsrno. of embryos cultured=100.
b
EB: early, MB: mid, EX: expanded, HB: hatched blastocysts.
c,d,e
P - 0.05,
f,g
P - 0.05.
SAS. Differences in preimplantation development of DNA-injected embryos between the two culture systems were analyzed by Student’s t-test in SAS.
3. Results
3.1. Experiment 1 To examine if MEF affect the in vitro development of IVF-derived bovine embryos,
663 immature oocytes were matured, fertilized and cultured in vitro. Out of 498 cleaved Ž
. embryos cleavage rate: 75.1, 498r663 at Day 3 of culture, only 4- to 8-cell stage
Ž .
embryos were selected and cultured further with or without MEF Table 1 . Develop- Ž
. mental rate to blastocyst of the embryos co-cultured with MEF 24.4, 49r201 was
Ž .
Ž significantly higher
P - 0.05 than that of embryos grown without MEF 14.4,
. 28r194 . In both culture systems, 8-cell embryos showed a higher developmental rate
Ž .
than 4-cell embryos P - 0.05 . Moreover, developmental rate to hatching or hatched
Table 2 Comparison of the in vitro development of DNA-injected embryos co-cultured with or without MEF
Group No. of embryos
No. of embryos No. of embryos
No. of embryos
a b
Ž . Ž .
injected survived
cultured that developed
c
Ž . to blasotcysts
U
Ž .
Ž .
Ž .
CR1aa 2931
2505 85.5 1804 72.0
131 7.2
U
Ž .
Ž .
Ž .
MEF 3370
2516 74.6 1805 71.7
302 16.7
a
No. of embryos survivedrno. of embryos injected=100.
b
No. of embryos culturedrno. of embryos survived=100.
c
No. of blastocystsrno. of embryos cultured=100.
U
P - 0.01.
Table 3 Pregnancy rates after transfer of DNA-injected bovine embryos that developed in CR1aa medium with or
without MEF
a
Ž . Group
No. of embryos transferred Pregnantrrecipients
No. of calves that developed to
b
Ž . Abortionrstill-born
Live-born Ž
. Ž
. CR1aa
93 16r66 24.2
3r4 9 56.2
Ž .
Ž .
MEF 131
23r84 27.4 2r2
19 82.6
a
Ž . means the pregnancy rate: pregnantrrecipients=100.
b
Ž . : No. of calves live-bornrpregnant recipients=100.
Ž .
blastocysts of IVF-derived embryos was higher P - 0.05 in the co-culture group
Ž .
Ž .
10.9, 22r201 than in CR1aa medium alone 4.1, 8r194 . 3.2. Experiment 2
Ž .
Although there was no difference in the survival 85.5 vs. 74.6 and cleavage Ž
. rates 72.0 vs. 71.7 of DNA-injected zygotes between the two different culture
systems, the developmental rate to blastocyst stage of DNA-injected embryos co-cul- Ž
. tured with MEF 16.7, 302r1805 was significantly higher than that of the embryos
Ž .
cultured in CR1aa medium alone 7.2, 131r1804; P - 0.01, Table 2 . 3.3. Experiment 3
When DNA-injected bovine blastocysts co-cultured with MEF were transferred to Ž
. recipients, the pregnancy rate 27.4, 23r84 was similar to that of the embryos grown
Ž .
in CR1aa medium without MEF 24.2, 16r66; Table 3 . However, the proportion of Ž
. live-born calves among was higher in the co-culture group 82.6, 19r23 than in
Ž .
CR1aa medium alone 56.2, 9r16 . Among calves developed from co-cultured
embryos, one male calf died at 1 day after birth. This calf did not suckle and no further investigations were made into its death. The other calves in both culture groups appeared
to be normal at birth and their subsequent growth was normal. We did not carry out any histopathological test on the aborted and still-born calves. Blood and ear tissue samples
of the calves were analyzed for the presence of a transgene by Southern blot, but no transgenic animals were identified.
4. Discussion
