. sponding to estimated time from activation display to start of the pronuclear stage ,
therefore the minimal number of pulses will be four. However, extending pulse sequence is likely to affect development. Thus, as the time sequence enlarges, oocytes age being
more sensitive to calcium stimulus, which would compromise their ability to develop to
Ž .
Ž .
blastocyst Stice and Robl, 1988; Collas and Robl, 1990 . Moreover, Kaufman 1978 indicated the detrimental effect that culture and handling conditions to which eggs were
submitted during the first hour following activation would exert upon mouse parthenote development. In our conditions, this fact may be aggravated by using a sub-optimal
Ž .
culture medium Brackett and Oliphant’s defined medium over longer activating time period.
Thus, the objectives of this work are to test the effects of extending four and eight pulse sequences upon activation rate, type and even subsequent parthenogenetic devel-
opment, in order to define an efficient activating procedure which would increase the haploid embryo production.
2. Materials and methods
2.1. Oocyte collection and preparation Mature mixed-breed female rabbits were tested for sexual receptivity and treated with
Ž .
25 IU of hCG Coriogan, Lab. Ovejero, Spain . The does were not superovulated. Oocytes were recovered from oviducts 14 h after endovenous hCG treatment by flushing
Ž .
in Dulbecco’s phosphate-buffered saline DPBS immediately after euthanasia of the donor females. The cumulus-enclosed oocytes were briefly placed in Ca
2q
-free DPBS Ž
supplemented with 1 mgrml bovine testes hyaluronidase Cat. No.: H4272. Type IV-S, .
Sigma, Spain , then gently pipetted through a small-bore pipette to free them from corona cells. After treatment, oocytes were selected for the presence of a clearly visible
Ž .
first polar body PB1 and for general healthy appearance, then cultured at 398C in Ž
. defined medium DM; Brackett and Oliphant, 1975 in a 7 CO
humidified atmo-
2
sphere. 2.2. Oocyte actiÕation
Oocyte activation was accomplished by electrical stimulation initiated at 15–15.5 h post-hCG. Handling, pulse parameters and pulsing medium characteristics have been
Ž .
described Escriba and Garcıa-Ximenez, 1999 . Briefly, oocytes were placed between
´ ´
´
two stainless-steel round wire electrodes approximately 0.5 mm apart and overlaid with 0.18 M mannitol containing 100 mM CaCl
and 100 mM MgCl . Oocytes were
2 2
electrically stimulated with four or eight square electrical DC pulses of 0.6 kVrcm for 60 ms each over 90, 150 and 270 min, at regular time.intervals. Pulses were delivered by
Ž .
an electro-cell porator Cruz et al., 1995; Garcıa-Ximenez et al., 1995 and monitored
´ ´
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
