Table 1 Effect of B2–BRL coculture vs. cell-free B2 with or without serum on blastocyst production and the
proportions of different stages of blastocysts on Day 8 of culture Media
B2 B2qserum
B2–BRL coculture
a b
c
Ž . Ž
. Ž
. Ž
. No. blastsroocytes
17r303 5.6 13r73 17.8
79r168 47.0
a b
b
Ž .
Ž .
Ž .
Ž .
No. EB of total blasts 9 52.9
2 15.4 4 5.1
a a
b
Ž .
Ž .
Ž .
No. MB 6 35.3
4 30.8 8 10.1
a b
b
Ž .
Ž .
Ž .
No. XB 2 11.8
7 53.8 32 40.5
Ž .
No. HB 35 44.3
a,b,c
Means in rows without common superscripts differ, P -0.05.
chi-square analysis. Differences between treatment in stage, grade the number of total cells and pyknotic cells per embryo was determined by ANOVA and separate means
were then analyzed by Tukey’s HSD test.
3. Results
As shown in Table 1, it is obvious from Experiment 1 that B2 medium, whether or not it was supplemented with 10 serum, did not support development of bovine
zygotes to the blastocyst stage as efficaciously as did B2 supplemented with 10 serum in a BRL coculture system. Not only did coculture produce a much higher percentage of
blastocysts from the oocytes that were placed into IVM, but a higher proportion of the blastocysts were more advanced in development than those in B2 either with or without
serum. In addition, blastocysts resulting from culture in B2, whether or not serum was present, were composed of significantly fewer cells and were, on average, graded as
Ž .
lower in quality than blastocysts that were produced in B2–BRL coculture Table 2 . Furthermore, embryos raised with serum, either with or without coculture, were com-
Table 2 Effect of B2–BRL coculture vs. cell-free B2 with or without serum on embryo stage of development, embryo
grade, percentage of pyknotic cells and total cell number Embryo characteristics
Media B2
B2qserum B2–BRL coculture
1 a
b c
Stage 2.67
3.50 4.26
a a
b
Grade 2.00
1.92 1.47
a b
b
Pyknotic 26.5
14.3 8.4
a a
b
No. cells 54
66 155
a,b,c
Means in rows without common superscripts differ, P -0.05.
1
Stages: EBs 2, MBs 3, XBs 4, HBs 5.
Table 3 Production of bovine blastocysts from slaughterhouse-derived oocytes cultured in B2–BRL coculture with or
without serum for the first 72 h of culture Ž .
Ž . B2–BRL coculture
No. oocytes No. cleaved
No. blastocysts Day 7
Day 8 Ž
. Ž
. Ž
. Serum
516 304 58.9
155 30.0 183 35.4
Ž .
Ž .
Ž .
Ž .
No serum D1–4 504
340 67.4 162 32.1
186 36.9
posed of a lower percentage of pyknotic cells compared to embryos produced in B2 without serum.
In Experiment 2, zygotes resulting from slaughterhouse-derived oocytes and cultured Ž
. in B2–BRL coculture without serum during Days 1–4 72 h developed into blastocysts
by Days 7 and 8 at the same rate as zygotes exposed to serum throughout the culture Ž
. period Table 3 . By Day 4 of culture, there was some sloughing of BRL cells from the
bottom of the culture wells. However, this was not accompanied by any observable influence on the percentage of oocyte cleavage or the rate of embryo development in
this treatment.
In Experiment 3, there was no difference among the three culture systems in the efficiency, which ranged from 17.8 to 19.6, of embryo production from the oocytes
Ž .
that were placed into IVM Table 4 . Also, there was no difference in 60-day pregnancy rates in recipient heifers resulting from the transfer of embryos produced in the three
different culture systems. The sex of embryos, as determined by three different methods Ž
. as described in Section 2 , was not different for embryos produced among the three
Ž .
different culture systems Table 4 . The percentage of males for each separate treatment Ž
. and for all treatments combined 54.2 did not differ significantly from a sex ratio of
50:50. There were no differences in the distribution of embryo stages produced in Ž
. B2–BRL coculture with or without serum Table 5 . However, B2 cocultures, either
Table 4 Embryo production and pregnancy rate following transfer of embryos derived from OPU-collected oocytes
cultured in B2–BRL coculture with or without serum for the first 72 h of culture or TCM 199–BRL coculture with serum
B2 TCM 199
Serum No serum
Serum No. oocytes
3250 3002
1725 No. embryos
638 535
313 Embryosroocytes
19.6 17.8
18.1
a
No. transfers 467
382 129
Ž . Ž
. Ž
. Ž
. No. pregnant
223 47.8 180 47.1
65 50.4
b
Ž . Ž
. Ž
. Ž
. No. males
186r345 53.9 152r280 54.3
36r65 55.4
a
Does not include embryos that were biopsied or frozen.
b
Includes biopsied embryos, ultrasounded pregnancies and calves.
Table 5 Distribution of different stages of embryos derived from OPU-collected oocytes cultured in B2–BRL coculture
with or without serum for the first 72 h of culture or TCM 199–BRL coculture with serum Ž
. No. embryos total
B2 TCM 199
Serum No serum
Serum
a b
Ž .
Ž .
Ž .
Morulae 84 13.2
52 9.7 27 8.6
Ž .
Ž .
Ž .
Early blastocysts 186 27.2
149 27.8 95 30.4
a a
b
Ž .
Ž .
Ž .
Mid blastocysts 191 29.9
183 34.2 139 44.4
a a
b
Ž .
Ž .
Ž .
Expanded blastocysts 155 24.3
137 25.6 51 16.3
a a
b
Ž .
Ž .
Ž .
Hatched blastocysts 22 3.4
14 2.7 1 0.3
Total 638
535 313
a,b
Means in rows without common superscripts differ, P -0.05.
with or without serum, produced a higher percentage of expanded and hatched blasto- cysts than did the TCM 199–BRL coculture system.
There was no difference in the percentage of spontaneous abortions, live births or Ž
. dystocias among the three different coculture systems Table 6 . There was no way to
accurately differentiate between Caesarean deliveries that were scheduled prior to the onset of labor vs. those that were performed as the result of calving problems in
recipients. Nevertheless, there was no difference in the survival rate of calves following Caesarean delivery among the three coculture systems. There was, however, a signifi-
Ž .
cantly lower survival rate P - 0.05 following natural delivery of all calves combined Ž
. for the three coculture systems 187r228 s 82.0 compared to the total of all calves
Ž .
that survived Caesarean delivery 55r59 s 93.2 . There were congenital abnormalities in calves and cases of hydro-allantois observed in recipients that received embryos from
either of the two B2 coculture systems, but neither problem was observed in the
Table 6 Pregnancy and calving characteristics following transfer of embryos derived from OPU-collected oocytes
cultured in B2–BRL coculture with or without serum for the first 72 h of culture or TCM 199–BRL coculture with serum
B2 TCM 199
Serum No serum
Serum
a
Total no. pregnancies 273
201 65
Ž . Ž
. Ž
. Ž
. No. abortions
36 13.1 24 11.9
7 10.7 No. calvings
113 70
45 Ž .
Ž .
Ž .
Ž .
No. live calves 89 78.8
57 81.4 41 91.1
Ž . Ž
. Ž
. Ž
. No. dead calves
24 21.2 13 18.6
4 8.9 No. caesarians
26 21
12 Ž .
Ž .
Ž .
Ž .
No. live calves 23 88.5
20 95.2 12 100
Ž . Ž
. Ž
. No. dead calves
3 11.5 1 4.8
Ž . Ž
. Ž
. No. congenital malformations
4r139 2.9 2r94 2.1
Ž . Ž
. Ž
. No. hydro-allantois
2r273 0.7 1r201 0.5
a
Includes intact and biopsied embryos.
pregnancies resulting from embryos cultured in TCM 199. A frequently reported characteristic of the pregnancies resulting from all three culture systems was that labor
was not clearly pronounced in the recipients.
4. Discussion
