Ž .
with an oscilloscope TDS 320, Tektronix, Spain . Oocytes were transferred from mannitol to DM between pulses.
2.3. Embryo culture Ž
Following electrical treatment, oocytes were cultured in DM without CCB-diploidis- .
Ž .
ing treatment for 6–7 h and were assessed for second polar body PB2 extrusion Ž
. Onodera and Tsunoda, 1989; Ozil, 1990 . After final checking PB2 extrusion, the
Ž .
oocytes were cultured in Ham’s F-10 medium plus 20 vrv homologous-doe serum Ž
. s-Ham’s under a 7 CO
atmosphere at 398C. After the first 24 h of culture,
2
non-divided oocytes were removed from culture. Embryo development was checked Ž
. every 24 h until end of culture 5 days .
2.4. Assessment of actiÕation types The different degrees of activation were established by checking PB2 extrusion
Ž andror further cleavage. Three types of parthenogenetic eggs were produced Kaufman,
. 1978; Ozil, 1990; Collas et al., 1993; Escriba and Garcıa-Ximenez, 1999 . The first type
´ ´
´
had one pronucleus plus the first and second polar bodies and further cleavage Ž
. PB1 q PB2 and divided ; it conduces to an haploid condition and is named normal
activation. The second type had two or more pronuclei plus the first polar body and Ž
. cleaved PB1 and divided , named as abnormal activation which usually determines a
diploid condition. The third type had the first and second polar bodies but no subsequent Ž
. cleavage PB1 q PB2 and non-divided , named incomplete activation.
At least three replicates were performed per group. The results were analysed using a Chi-square test. When a single degree of freedom was involved, the Yate’s correction
for continuity was carried out.
3. Results
Activation results are summarised in Table 1. With four pulses, the activation Ž
frequency significantly increase when treatment duration was extended from 54 to .
100 . Regardless of treatment duration, eight pulses induced oocytes to activate Ž
. efficiently 91 to 97; P 0.05 . The effect of number of pulses is only statistically
significant at 90-min treatment duration: more pulses induced greater oocyte activation Ž
. eight: 92 vs. four: 54; P - 0.05 .
Ž The type of activation is also shown in Table 1. Incomplete activation PB1 q PB2
. and non-divided was present in all treatments at low frequency, following no specific
trend. Ž
. After four pulses, normal activation rates PB1 q PB2 and divided were not affected
Ž .
by treatment duration from 51 to 80; P 0.05 . However, haploid frequency was Ž
significantly enhanced by extending total eight-pulsing treatment duration 90 min: 47; .
150 min: 55 and 270 min: 76 . The normal activation rate was affected by the
Table 1 Effect of number of pulses and pulsing-sequence duration on rabbit oocyte activation
1 2
Ž . Number of
Treatment Number of oocytes
Type of activation pulses
duration Ž .
Pulsed Activated
PB1qPB2 and PB1qPB2 and
PB1 and Ž
. min
non-divided divided
divided
c a
a,b
Ž .
Ž . Ž
. Ž
. 90
37 20 54
0 0 16 80
4 20
b a,b
a,b
Ž .
Ž .
Ž .
Ž .
4 150
44 37 84
7 19 19 51
11 30
a a
b
Ž .
Ž . Ž
. Ž
. 270
46 46 100
2 4 34 74
10 22
a,b b
a
Ž .
Ž . Ž
. Ž
. 90
37 34 92
1 3 16 47
17 50
a,b a,b
a,b
Ž .
Ž . Ž
. Ž
. 8
150 46
42 91 1 2
23 55 18 43
a,b a
a,b
Ž .
Ž . Ž
. Ž
. 270
30 29 97
0 0 22 76
7 24
a – c
Ž .
Numbers with different superscripts within a column differ P -0.05 .
1
Three replicates per group were performed. Pulses of 0.6 kV cm
y1
lasting 60 ms were applied at regular time intervals.
2
PB1qPB2 and non-divided: oocytes with the first and second polar bodies and one pronucleus but no Ž
. Ž
. subsequent cleavage incomplete activation ; PB1qPB2 and divided: oocytes with PB2 one pronucleus and
Ž .
Ž further cleavage normal activation ; PB1 and divided: oocytes with suppressed extrusion of the PB2 two or
. Ž
. more pronuclei and further cleavage abnormal activation .
Ž number of applied pulses only when 90-min treatments were performed four pulses:
. 80 vs. eight pulses: 47; P - 0.05 .
Ž .
In abnormal activation rates PB1 and divided , significant differences were only Ž
. detected between the four-pulsed over 270 min group 22 and the eight-pulsed over
Ž .
90 min group 50 . Parthenogenetic embryo development is shown in Table 2 In normally-activated
Ž .
oocytes haploid condition , the rate of compacted morula was significantly increased Ž
. from 69 to 94 when the total duration of four-pulse treatment was increased but,
with the same number of pulses, the haploid-derived blastocyst rate was not affected by Ž
. treatment duration ranging from 15 to 37; P 0.05 . After applying eight pulses, a
significant contrary effect due to increase treatment duration was observed on haploid-
Table 2 Effect of number of pulses and pulsing-sequence duration on the subsequent in vitro development of haploid
and diploid parthenogenetic eggs
1 1
PB1qPB2 and divided PB1 and divided
Number of Treatment Number Number
Number Number Number
Number Ž
. Ž .
Ž . pulses
duration min cultured compacted
blastocysts cultured compacted
blastocysts Ž .
Ž . morulae
morulae
b,c a,b
a,b
Ž .
Ž .
Ž .
Ž .
90 16
11 69 5 31
4 4 100
2 50
a,b a,b
a,b
Ž .
Ž .
Ž .
Ž .
4 150
19 18 95
7 37 11
11 100 3 27
a a,b,c
a,b
Ž .
Ž .
Ž .
Ž .
270 34
32 94 5 15
10 9 90
2 20
a,b a,b
a
Ž .
Ž .
Ž .
Ž .
90 16
15 94 5 31
17 14 82
11 65
a,b,c b
b
Ž .
Ž .
Ž .
Ž .
8 150
23 17 74
6 26 18
15 83 3 17
c c
a,b
Ž .
Ž . Ž
. Ž
. 270
22 9 41
0 0 7
6 86 2 29
a – c
Ž .
Numbers with different superscripts within a column differ significantly P -0.05 .
1
See footnote, Table 1.
Ž .
Ž derived morulae rates from 94 to 41 and also in the blastocyst rates from 31 to
. 0; P - 0.05 .
Ž .
Diploid embryos derived from abnormally activated oocytes PB1 and divided did not show in vitro developmental differences up to compacted morula among the applied
Ž .
treatments, ranging from 82 to 100 Table 2; P 0.05 . Moreover, during in vitro diploid development up to the blastocyst stage, no trend has clearly emerged from either
the number of applied pulses or the total duration of the sequence. However, from all assayed combinations, only after applying eight pulse treatment over 90 min was the
Ž .
diploid in vitro development to blastocyst significantly higher 65 than that observed Ž
. after eight pulses over 150 min 17 .
Global data from every treatment were retrospectively grouped and showed that diploid eggs, exclusively obtained by PB2 retention but not by diploidising treatments,
Ž .
are able to develop to the morula and blastocyst stages, reaching an 88 59r67 and a Ž
. 34 23r67 , respectively. However, global haploid development up to the morula and
Ž .
blastocyst stage was slightly lower 78; 102r130 and 21; 28r130, respectively . Ž
. The highest global haploid compacted morula rate per pulsed oocytes 70: 32r46 was
obtained after four-pulse over 270-min treatment. These data were not shown in the tables.
4. Discussion
