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
Ž .
This work confirms that in short-length activating procedures 90 min , a higher Ž
. number of pulses here: eight improved the activation rates of young rabbit oocytes
Ž .
Collas and Robl, 1990; Ozil, 1990; Collas et al., 1995 but induced lower rates of Ž
. normal activation haploids: Escriba and Garcıa-Ximenez, 1999; this work .
´ ´
´
Ž .
After applying four pulses, extending sequence duration from 90 to 150 or 270 min improved global activation rates without penalising normal activation frequency. This
effect would be due to either enhanced oocyte sensitivity from their in vitro ageing ŽStice and Robl, 1988; Ware et al., 1989; Collas and Robl, 1990; Fissore and Robl,
. 1992; Collas et al., 1993; Prochazka et al., 1993; Adenot et al., 1997 , or to inhibition of
´
Ž MPF re-activation over a longer time period Fissore and Robl, 1992; Vitullo and Ozil,
. 1992 . Effectively, one electrical pulse promotes intracellular calcium rise which, after
Ž reaching a critical amplitude level, reduces the MPF activity temporarily Fissore and
. Robl, 1992; Collas et al., 1993, 1995 . In rabbits, this transient reduction does usually
Ž .
Ž spend 90 min Collas et al., 1995 . Thus, repetitive pulsing at every 90 min as with
. four-pulse and 270-min sequences would maintain the MPF activity at basal levels
Ž which is required for oocyte progression into early interphase stages Kubiak, 1989;
. Fissore and Robl, 1992; Collas et al., 1995; Wu et al., 1998 .
After applying eight pulses, extent of sequence duration increased haploid egg production by increasing normal activation rates. Within a pulsing sequence, when a
pulse becomes efficient, the meiosis is resumed and, following a certain time period, Ž
culminates with the PB2 extrusion following 90 min in rabbits: Oh and Brackett, 1975; .
Ozil, 1990 . During that period, application of electrical pulses, and their characteristics, Ž
will determine the final oocyte activation type Ozil, 1990; Collas et al., 1993;
. Prochazka et al., 1993; Escriba and Garcıa-Ximenez, 1999 . The PB2 retention due to
´ ´
´ ´
the over-stimulation applied over said critical period is more frequent the greater the Ž
pulsing frequency number of pulsesrtotal treatment duration; Ozil, 1990; Escriba and
´
. Garcıa-Ximenez, 1999 . This fact would explain why in eight-pulse sequence, a lower
´ ´
pulsing frequency, determined by long-length sequences, does not modify the global activation rates, which are always elevated, but increases the normal activation ones
Ž .
lower over-stimulation level . In earlier reports, following either 8- or 12-pulse se- Ž
. quences applied over 90 min high pulsing frequency , the global activation rates were
maximal, whereas normal activation rates were reduced or zero, and some oocytes even Ž
. lysed Escriba and Garcıa-Ximenez, 1999 .
´ ´
´
Parthenogenetic rabbit embryo development is usually referred to in diploid embryos ŽOnodera and Tsunoda, 1989; Ozil, 1990; Pinto-Correia et al., 1995; Mitalipov et al.,
. 1999 . However, in our knowledge, few works on haploid embryo development have
Ž .
been reported for this species Escriba and Garcıa-Ximenez, 1999; this work . In the
´ ´
´
present work, the in vitro embryo developmental ability of both haploid and diploid parthenogenotes was verified, obtaining high developmental rates to the compacted
Ž .
morula stage for diploids suppression of PB2-derived , and slightly lower for haploids. Previous reports have indicated that insufficient, or, conversely, excessive activation
Ž stimulus impairs parthenogenetic diploid embryo development Ozil, 1990; Collas et al.,
. 1995; Pinto-Correia et al., 1995 . In our work, both these types of detrimental effects
were detected, but only on haploid development. This would be due to different diploid Ž
. origin in our work derived from oocytes with the PB2 non-extruded and those used by
Ž .
other authors in all cases, diploidised by CCB-treatment . Haploid embryo development was impaired after applying the weakest assayed
Ž .
treatment four pulses over 90 min . Extension of four-pulse sequences improved
activation rates and further development. In contrast, after applying an eight-pulse sequence distributed over 270 min, the haploid embryo development was limited, being
evident at the morula, and even at the blastocyst stage. This would probably be due to the culture and handling conditions to which eggs were submitted during the first hour
Ž following activation, with the osmolarity of the media exerting a main effect Kaufman,
. Ž
. 1978 , and even the initial sub-optimal culture medium defined medium . Moreover, in
eight-pulse and 270-min sequences, more pulses would have been performed over the post-activation stage, when the oocyte is already at the interphase. In normal fertilisation
process, calcium-stimulus amplitude decreases as fertilisation progresses, not being
Ž .
detected at the pronuclear apposition stage Fissore and Robl, 1993 . Perhaps a higher number of calcium stimuli, applied over the pronuclear stage, would be responsible for
observed penalisation on subsequent embryo development. In fact, excessively high intracellular calcium levels have been shown to be detrimental to certain cell functions
Ž .
or structures Izant, 1983 . Whether these post-activating pulses are required or not for further parthenogenetic development will be the subject of subsequent research.
5. Conclusions
